Vol.12_No.2 - Pesticide Alternatives Lab - Michigan State University

Resistant Pest Management NewsletterA Biannual Newsletter of the Center for Integrated Plant Systems (CIPS) in Cooperation with the InsecticideResistance Action Committee (IRAC) and the Western Regional Coordinating Committee (WRCC-60)Vol. 12, No. 2 (Spring 2003)Table of ContentsLetter from the Editors 3Resistance Management ReviewsManaging Phosphine Resistance in Grain Insects with the Phoscard® - R. Emery 5International QoI Working Group of FRAC: Detection of Resistance to QoI Fungicides inSeptoria tritici in Wheat in Europe - Fungicide Resistance Action Committee 7Insecticide Resistance Action Committee - Current Overview - A. Porter and G. D.Thompson 8Historical Perspectives on Insecticide Resistance Research: New Study InvestigatesDevelopments in Scientific Approaches from First Known Cases to the PresentSituation - J. S. Ceccatti 9Resistance Management from Around the GlobeBaseline ResistanceNative Resistance to Cry1Ac toxin in Cotton Bollworm, Helicoverpa armigera (Hubner)in South Indian Cotton Ecosystem - B. Fakrudin, Badariprasad, K. B. Krishnareddy,S. H. Prakash, B. V. Patil, and M. S. Kuruvinashetti 10Insecticide Resistance in Cotton Bollworm, Helicoverpa armigera (Hubner) in SouthIndian Cotton Ecosystems - B. Fakrudin, Badariprasad, K. B. Krishnareddy, S. H.Prakash, Vijaykumar, B.V. Patil, and M. S. Kuruvinashetti 13Development of Resistance in Insects to Transgenic Plants with Bacillus thuringiensisGenes: Current Status and Management Strategies - S. V. S. Gopalaswamy, G. V.Subbaratnam, and H. C. Sharma 16Generating Baseline Data for Insecticide Resistance Monitoring in Cotton Aphid, Aphisgossypii Glover - P. M. Praveen and A. Regupathy 26Baseline Susceptibility and Quantification of Resistance in Plutella xylostella (L.) toSpinosad - R. K. Arora 27Baseline Susceptibility of Diamondback Moth, Plutella xylostella (Linn.) to NewInsecticides – B. S. Joia, K. S. Suri, and A. S. Udeaan 30Arthropod ResistanceResurgence of Spider Mite Tetranychus ludeni Zacher (Acarina: Tetranychidae) AgainstAcaricides and Botanical Pesticides on Cowpea - S. Kumar, S. Prasad, and R. N.Singh 32Insecticide Usage Patterns in South Indian Cotton Ecosystems to Control CottonBollworm, Helicoverpa armigera - B. Fakrudin, B. V. Patil, P. R. Badari Prasad, andS. H. Prakash 35Recent Advances in Host Plant Resistance to Whiteflies in Cassava - A. Bellotti, B. Arias,A. Bohorquez, J. Vargas, H. L. Vargas, G. Trujillp, C. Mba, M. C. Duque, and J.Tohme 38Evidence for Multiple Mechanisms of Resistance to Cry1Ac and Cry2A Toxins fromBacillus thuringiensis in Heliothis virescens - J. L. Jurat-Fuentes, F. L. Gould, and M.J. Adang 42Monitoring Onion Thrips Resistance to Pyrethroids in New York - A. M. Shelton, B. A.Nault, J. Plate, and J. Z. Zhao 44

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Letter from the EditorIn 2001 the world's pesticide market exceeded $34billion while in the US it is projected to have exceeded$11 billion. It has been estimated by Pimentel et al.(2002) that pesticide resistance surpasses $1.4 billionin environmental, ecological, and human impact costs.What are some of the features driving resistancedevelopment in North American societies?Consumerism certainly drives much of the globalismand free-market decisions in North America today. Bilateraltrade agreements and falling tariffs have openedthe way to new markets and products. Both pesticideregulations and the enactment of the Food QualityProtection Act (1996) are seen by some as emergentproperties of consumerism and the environmentalmovement. Consumers demand blemish-free fruit andvegetables. Federal and state regulations requirewholesome and labeled products as well as numerousother quality-related characteristics. Thus,consumerism in its myriad forms has swiftly overtakenoutdated forms of production, marketing, and sales ofagricultural products. Consumers have power in themarket place today, and their power is partiallytranslated into increased pressure toward "perfect"product quality that can only be delivered throughincreasingly intense pest management systems.The environmental movement has also fosterednew awareness and a drive toward new legislation andregulations targeting pesticides in agriculture andhealth protection. Environmental concern has also beenlinked to the consumer movement in western societiesand together they are global in scope, extending eveninto third world countries. Environmentalism transectsthe demographics of western societies and stronglyaffects the regulatory policies in the U.S., Canada, andMexico. It is projected that environmentalism willextend well into the twenty-first century.Environmentalism and consumerism together haveseveral pest management and resistance managementimpacts. The rise of global marketing, consumerism,and environmentalism together medicate much ofwestern society's modern conscience. First, NorthAmerican societies source products globally andtransport these goods rapidly into the country. Second,more than 60% of North American pests historicallyhave been introduced, with new introductionsoccurring almost weekly. At this rate, will NorthAmerican societies eventually import most of theecologically compatible global pest species despite ourphytosanitary barriers?The Scope of Resistance in North AmericaEmerging with consumerism on a global scale,market access through non-tariff phytosanitationbarriers has become a gauntlet that every entrepreneurmust overcome. Both the introduction of invasivespecies and phytosanitation requirements dictateadditional pesticide applications and potentiallyaccelerate resistance selection.Within this context is resistance, where thegenetic-based adaptation of pests to man's effort tocontrol them has become more and more important asglobalism, consumerism, and market access concernsdrive pesticide use. From this point of view, it is notdifficult to believe that resistance problems will plagueagriculture and human and animal health protection forthe foreseeable future.As editors, we represent applied ecology andinsect toxicology. In our view, it is difficult to lookpast the inference that resistance is a symptom of adysfunctional ecosystem. That is, agriculturalproduction systems are often defined as disruptedecosystems (Southwood, 1978). Resistance canlogically be viewed as a symptom or indicator of anecosystem that has been disrupted beyond its naturalequilibrium, resulting in an ecologically negativeoutcome. Therefore, resistance is a consequence ofpesticide overuse, or selection susceptibility genes in autilitarian and reductionist sense.This perspective could also be adopted in humanand animal health protection where the problem withanti-microbial resistance has surfaced in the popularmedia repeatedly. It is with some irony that mediawould focus on antibiotic resistance and human healthwhile less immediate resistance issues withinsecticides, herbicides, and fungicides in foodproduction rarely surface. Insecticide, acaracide, andfilaricide resistance is also a critical issue for humanhealth protection in North America but the mediasurprisingly overlooks it too. The media even ignoresefforts by the US Centers for Disease Control (CDC)and the World Health Organization (WHO) to trackvarious disease vector resistance development in theAmericas, Asia, and Africa. With the recent mediaattention in North America on the introduction of themosquito-vectored West Nile Virus into suburban andurban population centers, one might expect a somewhatbroader articulation of the fragile nature of humanhealth protection, including vector resistance. A furtherirony, some might note, is that North American mediain concert with environmental and consumermovements would skewer certain insecticide use like3

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2organophosphates in food production, yet approve - oreven champion - the direct exposure of large numbersof people during mosquito vector control operations.Apparently, it is not appropriate to expose people tominuscule residues in the diet, but inhalation andcontact exposure for human health protection is notnewsworthy.When addressing the scope of North Americanresistance development, new regulations dealing withresistance are of critical interest. For example, with thepromulgation of regulations governing the registrationof genetically modified plants containing insecticidalproteins, resistance management plans were required asa prominent portion of the registration portfolio. Withone exception, all of the current conditionalregistrations for genetically modified plants containinginsecticidal proteins have a resistance managementplan based on high dose and refugia strategies (thesingle exception is Mom 863 for corn rootwormcontrol).The European Union has also recently taken somerecent strides to require resistance managementguidelines in its regulatory system. The EU-EPPO-PP1/213(1) guidelines require resistance riskassessment, development, and implementation of aresistance management plan and baseline monitoring ofresistance for all new registrations within the EU. The1996 Food Quality Protection Act (FQPA) also has aprovision for resistance monitoring contained in itsdetails. Essentially this prescription for resistancemonitoring is worded much like a series ofrecommendations of the US Board on Agriculture ofthe National Research Council, one of which statesthat, "Federal agencies should support and participatein the establishment and maintenance of a permanentrepository of clearly documented cases of resistance"(Dover and Croft, 1986). However, to our knowledgeno divisional program within USEPA has everfollowed up on this part of the FQPA law other thanvoluntary reporting of resistance development byregistrants.Presumably one measure of the impact of recentregulations on the availability of resistancemanagement tools is the number of differentformulations, pesticide and biopesticide modes ofaction, effective natural enemies, and othermanagement strategies, tactics, and tools.Approximately 6,000 pesticides have been cancelled orsignificantly mitigated since the passage of the FQPA.On the other hand, the FQPA and related activities ofthe USEPA have accelerated the registration ofreduced-risk pesticides and organophosphatealternatives. Unfortunately however, this legislationhas also practically eliminated the experimental usepermit process whereby land grant universities, privatetechnical service providers, and commodity researchershave historically adapted new pesticide tools to variousproduction systems. In addition, the FQPA hasprovided an array of new risk-science developmentsestimating the aggregate exposure to pesticides thatexhibit common modes of action, the cumulativehuman pesticide exposure over a lifetime, and theimpact of endocrine disruption on non-targetorganisms. Potentially all of these risk-scienceinnovations could have unique or integrated impacts onresistance and resistance management in NorthAmerica as the USEPA evolves these policies.As previously mentioned, resistance is a geneticbaseddecrease in the susceptibility of a population to acontrol measure. It has been observed acrossherbicides, fungicides, and bactericides, as well asinsecticides and miticides. An array of evolving pestbiotypes or races has also overcome conventionallyselected host plant resistance crop varieties. Perhapseven cultural control strategies like crop rotation maybe overcome by genetic adaptation in a pest. An arrayof adapted ecosystems, particularly resistant soils, hasalso evolved to pesticides. The economic, social, andenvironmental consequences for the various types ofresistance include pest control failures, disrupted pestmanagement systems (including limitations in thedevelopment of integrated pest management options),and increased pest control costs. Increased pest controlcosts have variously been classified as 1) pestmanagers forced to resort to newer, higher-pricedpesticide alternatives and 2) additional applications.Certainly, there are arrays of environmental,social, and disrupted functional ecosystemconsequences of increased pesticide use induced byresistance. Functionally, disrupted ecosystems andenvironmental impacts could be measured in increasedoff-target effects on bio-diversity and/or endangeredspecies. Additional social impacts may includeconsequences on humans from increased pesticideresidues, worker exposure, or increased disease spreadwhere vector control is diminished as a result ofresistance.In summary, globalism and environmentalism willlikely continue to impact the availability of pesticidesas well as the social and economic determinants thatwill dictate overuse of pesticides leading to resistance.Heightened concerns over homeland security,particularly in the United States, may have collateraleffects in terms of fighting bio-terrorism withadditional pesticide use. Certainly the emergence ofbiotechnology and genetically modified organisms withvarious pest selection processes could result in furtherexpansion of resistance problems. On the other hand,monitoring and diagnostics in resistance managementshould improve dramatically with the application ofnew high-through-put technology developed initiallyfor HIV/AIDS and cancer detection. In addition, thepesticide industry, though market and regulatoryincentives, is beginning to deliver an expanding array4

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2of novel and ecologically softer pesticides. This freshcollection of new modes of pesticide action shouldallow pest managers a greater diversity of managementtools to focus on target pests, thereby reducing the rateof resistance selection. Obviously the dissemination ofvarious regulations will continue to impact theavailability of resistance management tools.Certainly, society is witnessing the rapid andexpansive response of the private sector to reduced-riskand organophosphate-alternative incentives through theUSEPA. One might only speculate on the developmentof new resistance management strategies, tactics, andtools if some of the focus and resources currentlyemployed to regulate pesticides in North Americansocieties are actually allocated to monitoring andmeasuring resistance, the loss of susceptibility inresistant-prone species, or the dysfunctionalecosystems resulting from resistance development. TheCAST Resistance Conference highlighted severalefforts to document resistance development in weeds,fungicides, and arthropods. These efforts are essentialfrom our prospective, because "what gets measuredgets managed."Robert M. Hollingworth &Mark E. WhalonCenter for Integrated Plant SystemsMichigan State UniversityEast Lansing, MI 48824-1311USAResistance Management ReviewsManaging Phosphine Resistance in Grain Insects with the Phoscard®Export grain is one of the mainstays of Australianagriculture with over 80% of grain produced byAustralian growers destined for export. Unfortunatelythe warm storage conditions in Australia are conduciveto the establishment and development of grain insects.Australia's enviable record as an exporter of cleangrain has been achieved through the judicious use ofpesticides, fumigants, and general storage hygiene.More recently our customers have begun to demandgrain that is free from chemical residues as well asfrom insects and this has created challenges for graingrowers, handlers, marketers, and researchers.Australia is largely satisfying these markets forresidue-free grain with the use of fumigants,particularly phosphine. In Western Australia (W.A.)sealed storages are used on over 60% of farms and asimilar percentage of Bulk Handling storages aresealed. Since 1990, all grain has been exported fromW.A. without the use of contact insecticides at anystage during storage.This reliance on phosphine at all stages of storageplaces a lot of pressure on a single fumigant,particularly with respect to resistance development.Worse still, there are few alternatives - the use of the"fall back" fumigant, methyl bromide, is soon to beheavily restricted or terminated completely.Researchers around the world have shown that theineffective use of phosphine in poorly sealed storagescan lead to resistance and eventually control failures. InRob EmeryEntomology BranchDepartment of AgricultureWestern Australiathe early 1980's highly resistant strains of lesser grainborer were found in Bangladesh where phosphine hadbeen used for many years.Early research in Western Australia has shown thatgrain insect resistance rarely develops in bulk storagesbecause the cost and return of fumigating largeamounts of grain is so high that bulk handlers makesure the job is done correctly the first time, every time.In the past, resistance has developed on-farm wheregrain protectants and fumigants are not always used inaccordance with the label. There is a danger that thesestrains could find their way from the farm into thecentral handling system or worse still, an exportmarket.Bulk handlers routinely monitor the gasconcentrations in storages under fumigation throughoutthe fumigation period; this is the key missing factorfrom farm fumigations.Gas detection/monitoring equipment is often seenby farmers as being too expensive, too difficult tomaintain and calibrate, and too sensitive to the rigoursof day-to-day farmer use.As an alternative to monitoring, the currentrecommendation is that farmers pressure test theirstorages to assess the gas-tightness of the structureprior to fumigation. If the storage is sufficiently wellsealed, we know that an effective fumigation will takeplace provided the storage is kept sealed for 7-10 days.Unfortunately, from a farmer perspective, this pressure5

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2testing bears little relationship to the actualfumigation that appears successful becauseall the adults are dead. The trap of course isthat the eggs and pupae will very likely havesurvived the fumigation and are waiting inthe wings to turn into adults and reinfest thegrain within days.With support from the Department ofAgriculture Grains Program and the GrainsResearch and Development Corporation, wedecided to develop an extension tool in theform of a farm fumigation card that wouldoutline key points for conducting a safe andeffective fumigation. The card would alsohave an indicator strip that would givefarmers a "no frills" assessment of thestandard of their fumigation.The fact that phosphine gas corrodescopper is well known, the label even warnsagainst using phosphine around copperelectronic components. Early quarantinefumigation manuals from the United Statesrecommend placing a shiny penny inside railwagons before fumigation to give anindication of success at the end.We began experimenting with variousforms of copper until we found one thatgave an obvious response after exposure tolethal concentrations of phosphine for atleast 7 days. Copper used in electroniccircuit boards gave the best results and couldbe readily applied as a strip to plastic card.We tested the cards in over 20 sealedand unsealed farm storages. The copper stripconsistently indicated successfulfumigations. Figures 1 and 2 show typicalresults for good and poor fumigationscarried out over 7 days.The Phoscard® is styled on a creditcard with the front side bearing 5 keyfumigation principles (Figure 3). Thebackside (Figure 4) has a copper strip that isprotected from tarnishing by a layer oftransparent adhesive tape that must be removed beforeuse. There is a hole to attach string for retrieval andinstructions for use.Farmers can place a Phoscard® in storage prior tofumigation. If the copper strip is exposed to sufficientphosphine concentrations for at least 7 days, the shinycopper strip will turn almost completely black. In fact,it could end up looking like the inside of a car exhaustpipe - evenly coated with a black sooty substance, andin some cases verdigris may develop giving the copperthe distinctive blue/green colour often seen on corrodedcopper pipes. There is a colour chart printed alongsidethe copper strip to allow easy comparison.If the strip has not turned black at the end of thefumigation something has gone wrong: usually a leakystorage or insufficient number of aluminium phosphidetablets being placed in the storage. Remember,fumigate the storage space not the grain, use the samedose whether the storage is full or empty, and regularlyinspect and replace rubber seals.The Phoscard® is an extension tool for farmers. Itwill not replace phosphine monitoring equipment andis not intended to be used by fumigators or bulkhandlers. However, we are hopeful that farmersexperiencing fumigation failures indicated by thePhoscard®, will seek advice on how to improve theirfumigations. The cards will introduce farmers to thevalue of fumigation monitoring and may even6

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2encourage them to purchase one of thedigital phosphine meters to more accuratelymonitor their fumigations.The cards can be stored over longperiods while the protective tape remains inplace, but they can only be used once.Production costs are about US$0.40 each foran order of 5,000. Over 15,000 Phoscards®have been produced by the Department ofAgriculture and Co-operative Bulk HandlingWestern Australia and distributed amongfarmers.Sample Phoscards® are available from:Rob EmerySenior EntomologistDepartment of Agriculture3 Baron-Hay CourtSouth PerthWestern Australia 6151Ph: 08 9368 3247Fax: 08 9368 3223remery@agric.wa.gov.auandErnestos KostasGrain ProtectionCo-operative Bulk Handling (WA)22 Delhi St, West PerthWestern Australia 6005Ph: 08 9237 9600Fax: 08 9322 3942ernestos.kostas@cbh.com.auInternational QoI Working Group of FRAC: Detection of Resistance to QoI Fungicides in Septoria tritici inWheat in EuropeSensitivity Monitoring for Septoria Leaf Spot (Septoriatritici) in Wheat in 2002Extensive monitoring programmes were carriedout BASF, Bayer, and Syngenta throughout the wheatgrowing areas of Europe in 2002 using both regionalmonitoring approaches and targeting analysis of strainsfrom high risk trial sites.Field performance across Europe was good underhigh disease pressure. However, in a few locations inSouth West Ireland disease control was lower thanexpected. This was associated with severe diseaseFungicide Resistance Action Committee (FRAC)Klaus GehmannSyngentainfections, which were accentuated by agronomicfactors coupled with adverse weather conditions. Inthese sites, resistant isolates were found.In the monitoring programmes the vast majority oftested isolates was sensitive across Europe. A lowfrequency of resistant isolates was detected at specificsites in the UK and to a lesser extent in Germany andFrance.The G143A mutation was identified for theindividual resistant isolates in 2002. In retrospectivePCR analysis of some isolates collected in 2001a verylow frequency of the G143A was found indicating thatthe 2002 observations are the result of an ongoing7

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2selection process. Also, in 2001the field performancehad been good.Due to the epidemiology of Septoria, the spread ofresistance is expected to be much slower than thatobserved for wheat powdery mildew. Nevertheless, it iscritical in order to maintain the effectiveness of QoIs tostrictly implement, in practice, the guidelines givenconferred a low resistance factor. Studies are inprogress in order to investigate the significance ofthese isolates under practical conditions.Guidelines for Using QoI Fungicides on Cereal Cropsin 20031. Apply QoI fungicides according to manufacturersrecommendations for the target disease (orcomplex) at the specific crop growth stageindicated. Effective disease management is acritical parameter in delaying the build-up ofresistant pathogen populations.2. Apply use rates recommended by the manufacturerin order to ensure solid disease control andresistance management. The FRAC QoI workinggroup is concerned with the trend towards theapplication of decreased dose rates.3. Apply a maximum of 2 QoI fungicide containingsprays per cereal crop. Limiting the number ofsprays is an important factor in delaying the buildupof resistant pathogen populations.4. Apply the QoI fungicide preventively or as earlyas possible in the disease cycle. Do not rely onlyon the curative potential of QoI fungicides.5. Apply QoI fungicides in mixtures to control cerealpathogens. At the rate chosen each mixing partneron its own has to provide effective disease control.Refer to manufacturers recommendations for rates.6. Split / reduced rate programmes, using repeatedapplications, which provide continuous selectionpressure, must not be used.INTRODUCTION The Insecticide Resistance ActionCommittee (IRAC) is one of the sponsors of the MSUResistant Pest Management Newsletter and it wasconsidered an appropriate time to publish an overviewarticle outlining the background and objectives ofIRAC and update readers with some of the ongoingcurrent activities.IRAC was formed in 1984 to provide acoordinated crop protection industry response toprevent or delay the development of resistance in insectand mite pests. The mission of IRAC is to facilitatecommunication and education on insecticide resistanceand to promote the development of resistancemanagement strategies in crop protection and vectorcontrol so as to maintain efficacy and supportsustainable agriculture and improved public health.The organization is currently implementingcomprehensive strategies to confront resistance througha range of activities. In terms of organizationalstructure, IRAC along with the other Resistance ActionCommittees is a task force or working group ofCropLife International and as such is recognized byThe Food and Agriculture Organization (FAO) and theWorld Health Organization (WHO) of the UnitedNations as an advisory body. The group's activities arecoordinated via the IRAC Executive Committee, IRACInternational and Country or Regional CommitteesInsecticide Resistance Action CommitteeCurrent OverviewAlan Porter and Gary D. ThompsonIRACwith the information disseminated through meetings,workshops, educational materials and the IRACWebsite (www.plantprotection.org/irac). Groups arecomprised primarily of key technical personnel fromthe agrochemical companies affiliated with CropLifethrough membership in the relevant NationalAssociations (ECPA, CropLife America etc).OVERVIEW of ACTIVITIES The IRAC groups are activelyinvolved and, on certain occasions, provide funding fora variety of resistance management projects around theworld. These are generally driven or coordinated by thelocal country group and in some cases a specificproject group is set up to lead and ultimately reportresults and findings into the public domain. Examplesof these have been the long term monitoring ofmosquitoes resistance in Mexico and the monitoring ofpyrethroid resistance of Helicoverpa armigera in WestAfrican cotton. A new project group was set uprecently within IRAC International investigatingcodling moth resistance in a number of countriesaround the world. Other activities focus on issuesrelating to education, communication, and regulatoryapprovals as well as providing expert technical support.These more general activities are wide ranging but canbe grouped under the following headings:8

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2The Resistance Database - IRAC carried out andpublished an international resistance survey a numberof years ago but this is gradually becoming out of date.The survey is now being replaced by a new databasebeing developed at Michigan State University andsponsored in part by IRAC. This is a major effort andwill be the subject of a separate article in the ResistantPest Management Newsletter.Resistance Monitoring Methods - Reliable data onresistance, rather than rumors or assumptions, is thecornerstone of successful resistance management. Keyto this is the availability of sound baseline data on thesusceptibility of the target pest to the toxicant. A largenumber of bioassay and biochemical tests areemployed to characterize resistance but are notnecessarily comparable because different parametersand criteria are used. IRAC has evaluated and validateda wide range of testing methods that are published andare also freely available on the IRAC website. Newmethods are being evaluated and added to the list allthe time.Regulatory Approvals and Support - IRAC (along withHRAC and FRAC) have taken a leading role as anexpert group providing industry responses to proposalsfrom regulatory bodies. For example there is now aregulatory requirement in the EU under Directive91/414/EEC for companies to provide an assessment ofthe potential risk of resistance being developed bytarget organisms and for management strategies to beintroduced to address such risks. The ResistanceAction Committees (RACs) have been instrumental indeveloping workable guidelines for companiesresulting in the publication of an official GuidanceDocument. Similarly the US Environmental ProtectionAgency and the Pest Management Regulatory Agencyof Canada have been developing a voluntary pesticideresistance management labeling scheme based on modeor target site of action on the pest. The RACs havebeen heavily involved in classifying pesticides intospecific groups and families to enable the scheme towork. Development has been carried out under theauspices of the North American Free Trade Associationand has resulted in the issue of a Pesticide Registration(PR) Notice in the US.Education and Communication - IRAC has alwaysbelieved that education and communication plays a keyrole in the global management of resistance and hastaken many steps over the years to provide resources toacademia, researchers, industry, and growers. A newproject being undertaken this year is to update theexisting IRAC Education Kit that comprised of a video,35 mm slides, and supporting documentation toconduct an introductory workshop. Details about thekit will be announced in later editions of the ResistantPest Management Newsletter. Most of the IRACCountry groups, as well as utilizing centrallydeveloped resources, have their own educationalprograms in place, tailored to meet their local needs.IRAC US for example publishes articles on a regularbasis in grower magazines while IRAC Brazil holdstraining workshops in different locations. Other IRACGroups such as Australia, South Africa, Spain, andIndia have similar initiatives ongoing.IRAC Website - The existing IRAC website has nowbeen on-line for four years and has become the mainhome for IRAC information. Data available on-lineincludes the Monitoring Methods, MOA classificationscheme, Project and Country Group updates, MeetingMinutes, Member Contact details, Useful Links, detailsof Published Articles, and copies of new postersrecently produced.CONCLUSION IRAC is proud of its contribution to theCrop Protection Industry by improving awareness andmanagement of resistance issues. For this to continue itrequires not only the cooperation and support ofmanufacturers, regulators, extension workers,consultants, sellers, and users, but also effectivecommunication and compromise between technical andcommercial departments of all companies marketingcrop protection products. IRAC, with the support ofmember companies, intends to continue its role infacilitating this process.Historical Perspectives on Insecticide Resistance Research - New Study Investigates Developments in ScientificApproaches from First Known Cases to the Present SituationThe case of insecticide resistance provides anilluminating window into the workings of scientificJohn S. CeccattiPost-Doctoral Research Associate, Historical SeminarUniversity of Basel, SwitzerlandChemical Heritage Foundation, 315 Chestnut StreetPhiladelphia, PA 19106United Statesresearch and the complex interactions betweendisciplines and institutions. Beginning with the first9

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2scientific publications of the 1910s and continuing tothe present, the resistance question has drawnresearchers from fields as diverse as populationgenetics and organic chemistry and in institutionalcontexts ranging from agricultural experiment stationsto industrial research laboratories. To be sure,insecticide resistance research has been primarily thedomain of economic entomology, yet both the ubiquityand complexity of the phenomenon have broughttogether researchers with diverse backgrounds andresearch agendas.Detailing this history is the goal of a new researchproject funded by the Swiss National ScienceFoundation conducted by Christian W. Simon,professor of history at the University of Basel, andJohn S. Ceccatti, a post-doctoral research associate onthe project. One of the central questions beingaddressed is how scientists have utilized fieldobservations, laboratory experiments, and theoreticalframeworks to develop explanations of insecticideresistance that were consistent with current biologicalthought. Another area of interest is to compare thevarious research approaches taken by scientists fromvarious disciplines and institutional settings.From the current scientific vantage, the ability ofinsects to develop resistance to insecticides is hardly acontested fact. But even into the 1940s and 1950s, thephenomenon of insecticide resistance continued tochallenge many deeply held convictions amongscientists. For many economic entomologists, forexample, insecticide resistance ran against the longstandingidea of the fixity of biological species innature. Resistance also contradicted a guiding principleof the insecticide industry that once a chemicalcompound was proven effective is would remain soindefinitely. Resistance is also a sticky concept in thegeneral public - and, by extension, those corporatemanagers, policy-makers, and other 'thought leaders'without scientific training. One need only look at ongoingdiscussions (and confusions) about the relatedissue of antibiotic resistance to see that general beliefsin technological 'fixes' to complex problems havestrong staying power.To write the history of insecticide resistanceresearch, the authors draw on a variety of sourcesranging from scientific journals, industry trademagazines, unpublished technical reports fromcompany archives, as well as personal communicationswith scientists currently or formerly involved with theresistance question. On this latter point, the authorswelcome additional input and any interested personscan contact the authors by email atjohn@conceptualresearch.com. A synopsis of theresearch findings will be presented in the next issue ofthe RPM Newsletter.Resistance Management from Around the GlobeBaseline Resistance InformationNative Resistance to Cry1Ac Toxin in Cotton Bollworm, Helicoverpa armigera (Hubner) in South Indian CottonEcosystemGenes from Bacillus thuringiensis coding forcrystal (Cry) toxins of the Cry1A group have beentransferred to and expressed in a number of crops inorder to confer resistance against lepidopteron insectpests (1,2,3). Bt transgenic cotton was cleared by theDepartment of Biotechnology, Government of India forcommercial cultivation for the year 2002, after longdebate and discussion. The primary target pest of thistechnology in India and several other countries is thecotton bollworm, Helicoverpa armigera (Hubner),which causes economic losses up to about Rs. 250billion in India (3,4). Lately, the problems of pestmanagement in cotton and other crops have beencompounded by the development of resistance toinsecticides in H. armigera (5). Outbreaks of H.armigera in south Indian cotton and pigeonpeaecosystems usually lead to severe socio-economicaldisturbances, including several reports of suicide byfarmers. Introduction of insect resistant transgeniccrops, especially Bt transgenics, are expected to be ofimmense value in management and effective control oflepidopteran pests with a significant reduction in theoverall use of insecticides. However, long-termexposure to Bt transgenic crops is likely to renderlepidopteran pests resistant to the Cry toxins due tocontinuous selection pressure (6). Moreover, with theintroduction of transgenic plants, expressing a Crytoxin under the influence of constitutive promoters islikely to hasten this process. The development ofresistance to Bt toxins can be quite distinct, dependingupon the species, selection regime, or geographicalorigin of the founder colony (7). Hence, initial surveysto assess the susceptibility of test insects to the Crytoxins will establish a baseline that can be used inmonitoring resistance development in future. We reportthe resistance of H. armigera to Cry1Ac toxin in 11distinct geographic populations representing the entiresouth Indian cotton ecosystem.10

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2The Cry1Ac protein was produced according tothe method in Albert et al. (8) from an E. coli straincontaining the hyper-expressivity recombinant plasmidvector pKK223, kindly provided by Daniel R. Zeigler,Ohio State University, USA. The toxin was purifiedfrom over expressing cells by sonication and extensivewashing with sodium bromide. Proteins werequantified according to Lowry et al. (9) and the toxinwas quantified by SDS-PAGE densitometry beforepreparing dilutions (ranging from 10 to 20000 fold) indistilled water (10). Forty percent of the proteinextracted the recombinant E. coli cultures were foundto comprise Cry1Ac toxin. LC50 values weredetermined for the toxin.Laboratory strains of H. armigera were establishedfrom those collected in cotton fields during thecropping season of 2001-02 from major cotton growingregions of the south Indian cotton ecosystem: Nagpurand Nanded (Maharastra); Guntur, Madhira, andNalgonda (Andhra Pradesh); Dharwad, Raichur, andMysore (Karnataka); Coimbatore, Madurai, andKovilpatti (Tamil Nadu). These 11 sampling locationsrepresent the cotton growing ecosystems of south India(Fig. 1). An insecticide susceptible H. armigeraobtained from ICRISAT, Patancheru, Hyderabad wasused as a baseline susceptible strain for comparison.Larvae were reared on a chickpea-based semi-syntheticdiet (11), individually in 32-well multicavity trays untilpupation. Moths were kept in glass jars at 270C ±10Cand 70% RH and fed with a 10% honey solution. Alayer of muslin cloth was placed on the inner surface ofthe jars for oviposition.Laboratory cultures were established for eachpopulation from 500-650 moths and reared to gethomogenous F1 populations before conductingbioassays. Bioassays were carried out in 32-wellmulticavity culture trays. Six-day-old juvenile larvae(ca: 30-40 mg) were tested, one per well, on cottonleaves dipped in different concentrations of the toxin.In all, 30 larvae in three replicates were tested for eachtreatment. Mortality was recorded daily for six days.All assays were repeated three times and pooled datawere subjected to statistical analysis. Assays wereperformed in the laboratory at conditions at 270C ±10C and 70% RH. Median lethal concentrations(LC50) presented in Table 1 were derived from logdose probit calculations (12) using the MLP 0.38statistical package (13).Cry1Ac protein was found to be toxic to allgeographic populations tested (Table 1). Theinsecticide susceptible ICRISAT laboratory strain wasthe most susceptible. Compared with the others,geographic populations from Nagpur, Nanded, Guntur,Nalgonda, Madhira, and Raichur were found tolerant tothe toxin. Mortality of different populations ispresented in Table 1. LC50 values for Cry1Ac rangedfrom 0.147 to 1.095 µg/ml. The fiducial limits (atP=0.95) of the probit assay data indicated that therewas a good deal of variability in response of differentpopulations to Cry1Ac. The Kovilpatti (Tamil Nadu;extreme southern most part of south Indian cottonecosystem) population was found to be as susceptibleas the laboratory strain for Cry1Ac. The Coimbatoreand Dharwad populations were similar to each other ata resistance factor (RF) of 1.5. Geographic populationsof Guntur and Nanded recorded the highest RF of 8.03and 8.42, respectively. The LC50 values of the testpopulations could be considered as the baselinesusceptibility LC50 values for these individualpopulations and could be used for monitoringresistance in the future.For resistance management programs to beeffective, monitoring, surveillance, and earlydetection of resistant phenotypes in the fieldpopulations are important pre-requisites in orderto initiate timely remedial measures and toevaluate the effectiveness of resistancemanagement strategies. Traditionally, log doseprobit assays and recently diagnostic dose assays,have been routinely used to monitor developmentof resistance to insecticides (3, 14, 15).Diagnostic or discriminatory dose assays arenormally employed to identify individuals in apopulation resistant to the toxin (16), whereas logdose probit assays are useful to assess the level ofresistance of a population as a ration over areference strain or a population, usually asusceptible check. Therefore, for monitoringresistance built up in a population, diagnosticdose assays and log dose probit assays are themost appropriate (10,16,17). The results of thepresent analysis, showing significant differencesin susceptibility to Cry1Ac toxin among11

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2geographical locations of the south Indian cottonecosystem, is consistent with the studies of Gujat et al.(8) in Maharastra and Karnataka. Geographicalvariation in susceptibility to Bt toxins was earlierreported for the related species H. virescens and H. zea(10).One of the important exercises in the success of Bttransgenics is to assess and monitor baseline resistantlevels in representative geographical populations of thetarget insect and to ensure that it does not cross thepresent values. It is obvious that this value would varyfor each location/area. Data shows that even before theuse of Cry1Ac transgenics, levels of resistance were8.4 fold in the Nanded population followed byfollowed levels of 8.03, 7.70, 7.13,and 6.80respectively for Guntur, Nalgonda, Madhira, andRaichur. It was as low as +1.131 fold in the Kovilpattipopulation located in the extreme south. This is hard toexplain. Even where Bt sprays are used to some extentas a component of Integrated Pest Management (IPM)programs carried out in Andhra Pradesh and TamilNadu by the state department agencies, RF values arenot indicative of a definite trend. Apparently, there issome relationship in slope and RF value indicative ofheterogeneity and levels of resistance, respectively.Heterogeneity within a geographical location isexpected due to migratory nature of the H. armigeraand lack of selection history for Cry1Ac toxin in thesegeographic populations. Inter-population variation isdifficult to explain in a species like Heliothis (19).Notably, variability for response to the Cry1Ac toxindoes exist in the target population, whether or notpreviously exposed to the toxin. Except for the Raichurdistrict where Bt constitutes for 9.03% of the totalinsecticides used (unpublished data), Bt sprays hardlyconstitute 0.1% of the total insecticides used on cottonin these districts.The introduction of Bt transgenic crops is animportant addition to the existing components ofIntegrated Pest Management. The technology isperceived to be effective and eco-friendly. However,much of its success will depend on the sustainedsusceptibility of the target pests to the Bt toxins used intransgenic crops. Bt transgenic crops, which expressCry1Ac, were found to cause 100% and 75-90%mortality in susceptible H. virescens and H. zearespectively, in the United States of America (20). Thesame level of expression caused less than 90%mortality of H. armigera and H. punctigera inAustralia (21), indicating that Helicoverpa speciesappear to have certain levels of tolerance to the Bttoxin Cry1Ac, whether or not previously exposed to Bttoxin, when compared with the Heliothis species. It isimportant to note that in this study as well, a fewindividuals of H. armigera in almost all the populationstested were found to survive even the highestconcentrations of Cry1Ac tested. This would suggestthat, under field conditions, tolerant individuals arelikely to persist despite high expression of the Cry1Atoxins and may subsequently contribute to the resistantgene pool. Daly (22) reported that transgenic cottonplants in Australia killed susceptible larvae early in theseason but the effect significantly declined later (95-100 days after sowing), when an increasing proportionof first instar larvae placed on transgenic leavessurvived to late instars. The studies on Bt cotton in theUSA and Australia have shown that Cry1Ac proteinproduction decreased over the growing season and thatthe bio-efficacy of the protein was reduced byinteraction with increasing levels of secondary plantmetabolites (23,24). Differential expression in planttissues may contribute toward a reduced efficacy of theBt transgenic crops. If proper resistance managementstrategies are not implemented, the efficacy of pestmanagement through Bt transgenic crops will beseriously diminished due to widespread developmentof resistance. Such strategies have not yet beendeveloped for the small farmer and predominantly nonirrigatedcotton growing systems found in India andelsewhere.ACKNOWLEDGEMENT We thank Dr. Daniel R. Zeigler,Ohio State University, US for providing the Cry1Acover expressing clone. This research work wassupported by the DBT, GOI project grants to BF.Thanks are also due to Dept. of Agril. Entomology,UAS Dharwad for providing infrastructure facilitiesand all those scientists who helped us during the visit12

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2to different locations across south India for H.armigera collection. We thank Dr. G.T.Gujar, IARI,New Delhi, for his useful suggestions during the studyand encouragement.REFERENCES:1. Schuler, T.H., Poppy, G.M., Kerry, B.R. and Denholm, I., Tibtech,1998, 16: 168-1752. Jouanin, L., Michel, B., Girard, C., Morrot,G. and Giband, M., PlantSci., 1998, 131: 1-113. Kranthi, K. R., Kranthi, S. and Wanjari., R. R., Intl. J. PestManagement, 2001, 47: 141-1454. Manjunatha, T.M., Bhatnagar, V.S., Pawar, C.S. and Sithanatham, S.,proceedings of the International Workshop on Biological control ofHeliothis, New Delhi, India, 1985, p. 197-2285. Armes, N.J., Jadhav, D.R. and Lonergan, P.A., proceedings of theworld cotton research conference-I, Brisbane, Australia, 14-17 Feb.,1994, pp.552-5336. Kranthi, K. R., Kranthi, S., Ali, S. and Banarjee, S.K., Curr. Sci., 2000,78(8):1001-10047. Heckel, D., Biol. and Tech., 1994, 4: 405-4088. Albert, Z.G., Pfister, R.M. and Dean, D. H., Gene 1990, 93, 49-549. Lowry, D. H., Roseborough, A. L. and Randall, R.J., J. Biol. Chem.,1951.193: 265-27510. Sims, S.R., Berberich, S.A., Nida, D. L., Segalini, L.L., Leach, J.N.,Ebert, C.C. and Fuchs, R.L., Crop Sci., 1996, 36:1212-121611. Armes, N.J., Bond, G.S. and Cooter, R.J., Natural Resource InstituteBulletin, 1992, 5712. Finney, D.J., Probit Analysis, 3rd edition, Cambridge UniversityPress, Cambridge, 1971, pp.31813. Ross, G.E.S., Maximum Likelihood Program: The numericalalgorithms Gr. Rothamsted Experiment Station, Harpenden, UK,197714. Forrester, N.W., Cahill, M., Bird, L.J. and Layland, J.K., Bulletin ofEntomol. res., 1993, No 1, 1-13215. Kranthi, K.R., Armes, N.J., Rao, N.G.V., Raj, S. and Sundaramurthy,V.T., Pesticide Sci., 1997, 50: 91-9816. Roush, R.T. and Miller, G.L., J. Econ. Entomol., 1986, 79:293-29817. Georghiou, G.P. and Taylor, C.E., Proceedings of the 15thInternational Congress of Entomology, Washington DC, 1976,pp.759-78518. Gujar, G.T., Archana Kumari, Vinay Kalia and Chandrashekar, K.,Curr. Sci., 2000, 78(8): 995-100119. Fitt, G.P., Ann.Rev. Entomol., 1989, 34: 17-5220. Mahaffey, J.S., Bradley, J.R. and Van Duyn, J.W., Proceedings of theBeltwide Cotton Conference, 1995, 4: 563-57121. Forrester, N and Pyke, B., Australian Cotton Grower, 1997, 18: 23-2322. Daly, J.C., Biocontrol Sci. Tech., 1994, 4: 563-57123. Daly, J.C. and Fitt, G.P., In world cotton research conference-I,Athens, Greece, 1998, pp.18224. Federici, B.A., California Agriculture, 1998, 52: 14-2025. Litchfield, D. H. and Wilcoxin, F., J. Pharmaco ExperimentalTherapy, 1949, 96: 99-103.B. Fakrudin*, Badariprasad, K. B. Krishnareddy, S. H.Prakash, B.V. Patil#, & M. S. KuruvinashettiDepartment of BiotechnologyUniversity of Agricultural Sciences DharwadKrishinagar, Dharwad - 580005, KarnatakaIndia#Professor and HeadDept. of Agricultural EntomologyCollege of AgricultureRaichur - 584101, KarnatakaIndia*CorrespondenceInsecticide Resistance in Cotton Bollworm, Helicoverpa armigera (Hubner) in South Indian Cotton EcosystemsCotton occupies only 5% of the total cultivablearea in India but consumes more than 55% of the totalinsecticides used in the country, accounting for about250 billion rupees (Kranthi et al., 2001). Plantprotection continues to rely heavily on chemicalpesticides, a not very viable, long-term strategy if onelooks at recent failures against cotton bollworms andseveral other crop pests. Large-scale failures to controlHelicoverpa armigera Hubner (Lepidoptera:Noctuidae) in the major cotton-growing region ofSouth India in 1987 have been traced to insecticideresistance (McCaffery et al., 1989). To combat theunprecedented Helicoverpa armigera pest pressure,many farmers in the region applied syntheticpyrethroid, endosulfan, or organophosphateinsecticides, sometimes as mixtures, at 2-3 daysintervals during critical periods. This resulted in over30 sprays (against the 8-10 recommended) during theseason (Rakila et al., 1995), but growers were unable toachieve effective control with any of the availableinsecticides.The phenomenon of resistance to insecticides inHelicoverpa armigera that surfaced under differentagro-ecosystems of South India is the major negativeside effect of the chemical control strategy.Identification of baseline resistance to each of theinsecticide used in the region to control bollwormwould be indispensable for formulating effective IRMstrategies.The larvae of Helicoverpa armigera (second tosixth instar) collected from 12 different geographicallocations of South India (Fig. 1) were reared in the13

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2laboratory on a semi-synthetic diet toget F1 homogeneous larvae forbioassays.Larvae of 30-40 mg were used forbioassays. Six insecticides viz.,monocrotophos, chlorpyriphos,Endosulfan, carbaryl, cypermethrin,and quinalphos that are extensivelyused to control bollworm in cottonecosystems of South India were usedto determine baseline resistance. Thetechnical grade chemicals of theseinsecticides were obtained from M/SDe Nocil Crop Protection Ltd., M/SSyngenta Company, and M/S AventisCrop Science. Differentconcentrations of these selectiveinsecticides were prepared inanalytical grade acetone and aHamilton microapplicator was used todeliver a 1.0 µl drop to the thoracicdorsum of each third instar larva. Thecontrol larvae were treated withacetone alone. The concentrations ofeach insecticide were varied to obtain20-80 % mortality. Immediately afterexpose to insecticide/acetone, each of30 larvae (for each insecticide) waskept individually in a 30 ml plasticcup with fresh artificial diet andmortality was assessed 72hr aftertreatment. The dose-mortalityregression was computed by usingMLP 3.08 software (Ross, 1987).Monocrotophos: The Nagpurpopulation recorded a maximumLD50 value to monocrotophos(13.690 µg/µL) followed by thepopulation from Nalgonda (7.291µg/µL), Nanded (7.275), Guntur(7.027), Mysore (6.12), and Dharwad(4.22). The lowest LD50 value wasobserved in the population fromKovilpatti (0.308) followed byMadurai (0.452 µg/µL) andCoimbatore (0.777). The resistanceratio (RR) against the ICRISATsusceptible strain was found to behighest for the population of Nagpur(68.5 fold) followed by Nalgonda(36.5), Nanded (36.4), Guntur (35.1),Mysore (30.6), and Dharwad (21.1).The least resistance ratio was observed in thepopulation of Kovilpatti (1.5) followed by Madurai(2.3) and Madhira (3.9) (Table 1).Endosulfan: The Nalgonda population recorded amaximum LD50 value to endosulfan (13.240 µg/µL)followed by the population from Guntur (13.155µg/µL). The lowest LD50 value was observed in thepopulation from Madurai (0740 µg/µL) followed by14

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Madurai (3.4) followed byKovilpatti (4.1) and Coimbatore(4.7) (Table 2).Quinalphos: Maximum resistance toquinalphos was observed in theNalgonda population (20.937µg/µL)followed by the Guntur population(12.564 µg/µL). Least resistancewas noticed in the Maduraipopulation (0.5 µg/µL). Highresistance to quinalphos wasrecorded by the population fromNalgonda (123.2 fold) followed bythe Guntur population (73.9 fold) asagainst the susceptible ICRISATpopulation (Table 3).Chlorpyriphos: Maximumresistance to chlorpyriphos wasrecorded in Guntur (11.038 µg/µL)followed by Nalgonda (9.480µg/µL). Minimum resistance wasobserved in the Madurai population(0.36 µg/µL) followed by theKovilpatti population (0.463) andCoimbatore (1.128). The resistanceratio against the ICRISATsusceptible strain was found to behighest for the population of Guntur(78.8 folds) followed by Nalgonda(67.7) and Dharwad (33.4). Theleast ratio was recorded in thepopulation from Madurai (2.6) andKovilpatti (3.3) (Table 4).Kovilpatti (0906) and Coimbatore (1.025). Theresistance ratio (RR) against the ICRISAT susceptiblestrain was found to be highest for the population ofNalgonda (60.2 fold) followed by Guntur (45.7). Theleast resistance ratio was observed in the population ofCypermethrin: The Raichurpopulation recorded a maximumLD50 value to cypermethrin (22.40µg/µL) followed by the populationfrom Guntur (10.92) and Nalgonda(9.984). The lowest LD50 value wasobserved in the population fromMadurai (0.143µg/µL) followed byKovilpatti (0.192) and Coimbatore(2.472). The resistance ratio (RR)against the ICRISAT susceptiblestrain was found to be highest forthe population of Raichur (379.7fold) followed by Guntur (185.0)and Nalgonda (169.2). The leastresistance ratio was observed in thepopulation of Madurai (2.4) followed by Kovilpatti(3.3) (Table 5).Carbaryl: The Raichur population recorded amaximum LD50 value to carbaryl (13.36 µg/µL)15

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2followed by population from Nalgonda (9.13 µg/µL),Guntur (8.73), Mysore (5.15), and Dharwad (4.84). Thelowest LD50 value was observed in the populationfrom Madurai (0.78µg/µL) followed by Kovilpatti(0.87), Coimbatore (1.35), and Madhira (1.12). Theresistance ratio (RR) against the ICRISAT susceptiblestrain was found to be highest for the population ofRaichur (66.8 fold) followed by Nalgonda (45.7),Guntur (43.6), Mysore (25.8), Dharwad (24.2), Nagpur(14.6), and Nanded (10.7). The least resistance ratiowas observed in the population of Madurai (3.9)followed by Kovilpatti and Madhira (Table 6).The general LD50 values recorded were far highercompared to the recommended dosages indicating theexistence of resistance as was reported earlier (Armeset al., 1992). However, the resistance drastically differsfrom location to location within the South Indiancotton ecosystems. The resistance levels in the Guntur,Nalgonda, and Raichur regions (heavy insecticideusage areas) are due to heavy dependence oninsecticides. This clearly explained that resistancelevels were proportionate with the usage of pesticides.The study conducted by Forrester (1990) also clearlyrevealed that resistance levels rose when pyrethroidswere used but fell significantly when they werewithheld. Thus, the pesticides were creating very highselection pressure for resistant genotypes. Thissuggests that indiscriminate use and heavy dependenceon pesticide will further complicate the alreadyworsened situation and hints at aiming for insecticideresistance management strategies.ACKNOWLEDGEMENT: We thank Dr. Daniel R. Zeigler,Ohio State University, US for providing the Cry1Acover expressing clone. This research work wassupported by the DBT, GOI project grants to BF.Thanks are also due to Dept. of Agril. Entomology,UAS Dharwad for providing infrastructure facilitiesand all those scientists who helped us during the visitto different locations across south India for H.armigera collection. We thank Dr. G.T.Gujar, IARI,New Delhi, for his useful suggestions during the studyand encouragement.REFERENCES:KRANTHI.K.R., JADHAV,D.R., WANJARI,R.R., SHAKHIR ALI,S.AND RUSSEL,D., 2001, Carbamate and organophosphateresistance in cotton pests in India. Bull.Ent. Res., 91:37-46.Mc CAFFEREY, A.R., KING, A.B.S., WALKER, A.J. AND EL-NAYIR, H., 1989, Pestic.Sci., 27:65-76.RAKILA, A. AND PADMANABHAN, N.R., 1995,Knowledge andfactor influencing pesticide use and frequency of plant protectionmeasures. Pestology, 19:9-12. ROSS, G.J.S., 1987,Maximumlikelihood programme. The Numerical Algorithm groupRothemsted Experimental Station, Harpendon,U.K.ARMES, N.J., JADHAV, D.R., BOND, G.R. AND KING, A.B.S., 1992,Insecticides resistance in Helicoverpa armigera in South India.Pestic Sci., 34:335-364.FORRESTER, N.W., 1990, Designing, Implementing and Servicing anInsecticide resistance Management Strategy. Pestic sci., 28:167-179.B. Fakrudin*, Badariprasad, K. B. Krishnareddy, S. H.Prakash, Vijaykumar, B.V. Patil#, & M. S.KuruvinashettiDepartment of BiotechnologyUniversity of Agricultural Sciences DharwadKrishinagar, Dharwad - 580005, KarnatakaIndia#Professor and HeadDept. of Agricultural EntomologyCollege of AgricultureRaichur - 584101, KarnatakaIndia*CorrespondenceDevelopment of Resistance in Insects to Transgenic Plants with Bacillus thuringiensis Genes: Current Status andManagement StrategiesINTRODUCTION There is a continuing need to increasefood production as the world population is expected toexceed 6 billion by 2050. In both the developed andundeveloped countries, the cost for achievingproduction has become too high because of the need toincur costs for controlling insect pests that cause anestimated loss of $10 billion annually. The difficultiesexperienced in controlling insect pests over the past 30years have been largely due to the over-use ofpesticides. Indiscriminate use of insecticides has ledboth to the development of resistance in insects and thedestruction of natural enemies. Bio-pesticides such asBt (Bacillus thuringiensis) products are widelyregarded as being the least harmful to natural enemies.Because of its selectivity and environmental safety,usage of Bt is increasing, particularly in IPM programs.Foliar application of Bt breaks down quickly underfield conditions due to UV sensitivity and rainfall.With the advent of recombinant DNA technologies,insecticidal proteins present in Bt have been expressedin crop plants to ensure durable insect resistance. Thereis a considerable increase in global area undertransgenic crops from 1.7 million hectares in 1996 to52.6 million hectares in 2001, in which the share of Btcrops was 15% of the total area (James 2001).Although much progress has been made in thediscovery of new genes for introduction into plants,only Bt genes have been exploited so far.Considerable efforts have been made toincorporate delta-endotoxins from Bt into cereals, rootcrops, leafy vegetables, forage crops, and horticulturalcrops (Schuler et al. 1998). Of the $8.1 billion (US16

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2dollars) spent annually on insecticides worldwide, itwas estimated that nearly $2.7 billion could besubstituted with Bt biotechnology applications(Krattiger 1997). Economic advantage gained during1999 by Bt cotton alone has been estimated to be $213million in the USA. Cultivation of transgenic crops hasled to a reduction in pesticide use and significantincrease in yield (Cannon 2000). Unfortunately, thereare also concerns that the benefits of geneticallytransformed plants will be short-lived (McGaughey &Whalon 1992). Despite the potential advantages ofusing Bt crops, the possibility of their widespread usehas raised some potential problems. Decades ofindiscriminate insecticide use have demonstrated thatexposing insect populations to high levels of toxinsresults in evolution of resistance to insecticides (Roush& McKenzie 1987). Recently, several species of insectpests have been selected for resistance to Bt in thelaboratory, indicating that biological pesticides cansuffer the same fate as the chemical pesticides (Lianget al. 2000, McGaughey et al. 1998a).DEVELOPMENT of RESISTANCE in INSECTS to Bt GENESSeveral studies have shown that insect pests can adaptto Bt toxins under laboratory conditions (Shelton et al.2002). Certain pests such as Plodia interpunctella(McGaughey 1985), Heliothis virescens (Stone et al.1989), Plutella xylostella (Tabashnik et al. 1990),Spodoptera exigua (Moar et al. 1995), and Ostrinianubilalis (Huang et al. 1997) have been shown todevelop some degree of resistance to B. thuringiensisunder laboratory conditions. Evolution of insectresistance to insecticidal proteins produced by Btwould decrease our ability to control agricultural pestswith genetically engineered crops designed to expressgenes coding for these proteins (Gould et al. 1992).Information on development of resistance in insects toBt toxins has been summarized below.Indian meal moth, Plodia interpunctella:The first studied case of resistance to Bt-strainswas P. interpunctella, which had developed 100-foldresistance following 15 generations of laboratoryselection with Dipel (McGaughey 1985). On furtherselection, after 36 generations, the resistance levelsreached 250-fold (McGaughey & Beeman 1988).Bacillus thuringiensis sub sp. kurstaki caused a narrowspectrum resistance to Cry1Ab and Cry1Ac toxins,while sub sp. aizawai and entomocidus strains causedbroad-spectrum resistance to Cry1Aa, Cry1Ab,Cry1Ac, Cry1B, Cry1C, and Cry2A (McGaughey &Johnson 1994).Diamondback moth, Plutella xylostellaAlthough there are no instances of insectsdeveloping resistance to Bt transgenic plants in thefield, diamondback moth, P. xylostella, is the firstinsect known to have evolved high levels of resistanceto Bt as a result of repeated use of formulated Btinsecticide (Tabashnik et al. 1990). A diamondbackmoth colony derived from field population in thePhilippines that was regularly exposed to Dipel showedmore than 200-fold resistance to Cry1Ab (Ferre et al.1991). As much as 1640-fold resistance to Bt has beenrecorded in localized populations of diamondbackmoth from Hawaii, Florida, and Asia (Tabashnik et al.1992). In field populations of P. xylostella, resistanceto Bt sub sp. kurstaki, containing Cry1A(a,b,c), Cry2A,and Cry2B toxins and to a lower extent Bt sub sp.aizawai, containing Cry1A (a,b), Cry1C, and Cry1Dtoxins has been observed in various countries(Tabashnik 1994). Laboratory selection of P. xylostellausing Cry1Ca protein and in later generation transgenicbroccoli expressing Cry1Ca, increased Cry1Caresistance to 12400-fold (Zhao et al. 2000b).Resistance to Cry1A toxins from Bt sub sp. kurstakicaused cross-resistance to Cry1F, but not to Cry1B orCry1C (Tabashnik et al. 1996). Contrary to theassumption that independent mutations are required tocounter each toxin in P. xylostella, an autosomalrecessive gene conferred extremely high resistance toCry1Aa, Cry1Ab, Cry1Ac, and Cry1F (Tabashnik et al.1997). In a P. xylostella colony possessing 1500-foldresistance to a commercial formulation, the resistancerapidly fell to 300-fold in the absence of selection, butremained stable at this level in subsequent generations(Tang et al. 1996).Cotton bollworm/ legume podborer, Heliothis/HelicoverpaHelicoverpa armigera is capable of developingresistance to Cry1Ac in 7 to 8 generations (Kranthi etal. 2000). Highly mobile polyphagous pests such asHelicoverpa may develop resistance to Bt on onetransgenic crop and then disperse, nullifying theeffectiveness of a wide range of Bt transgenic cropsexpressing the same or similar Cry proteins. Pests withresistance to CryIA proteins in transgenic plants mayalso display significant resistance to Bt biopesticides.A laboratory strain of H. virescens developedresistance in response to selection with the Bt toxinCryIAc. In contrast to other cases of Bt-toxinresistance, this strain exhibited cross-resistance to Bttoxins that differ significantly in structure and activity(Gould et al. 1992). Over 10000-fold resistance toCry1Ac was obtained in H. virescens colony onselection with Cry1Ac protoxin (Gould et al. 1995).The insecticidal activity of Bt in leaves and squares oftransgenic cotton plant was high during the secondgeneration of the insect, but declined in the third andfourth generations of H. armigera in North China. Thesurviving third and fourth generation larvae, afterfeeding on flowers of Bt cotton, fed on the bolls untilpupation, which caused selection in field populationsof H. armigera. The increase in resistance was 7.1-fold17

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2after 17 generations of selection in the laboratory(Zhao et al.1998). Liang et al. (2000) found that theresistance ratio of H. armigera to Bt transgenic cotton,after selection for 16 generations was 43.3, andinheritance of resistance was controlled by a singleautosomal incomplete recessive allele.European corn borer, Ostrinia nubilalisThere has been a significant decrease insusceptibility across generations for selected strains ofO. nubilalis after chronic exposure to formulatedCry1Ab (Huang et al. 1997, Josette et al. 2001).Similarly, a 162-fold increase in resistance totransgenic Cry1Ac has been observed in European cornborer after 8 generations of laboratory selection (Bolinet al. 1999). Event 176 Bt corn hybrids express highlevels of Cry1Ab toxin in green plant tissue and pollen,but extremely low levels in the silk and kernels (Kozielet al. 1993), on which second generation O. nubilalislarvae have been shown to survive (Siegfried et al.2001). Zoerb et al (2003) stated that successfullydeveloped O. nubilalis larvae have either survivedexposure to sublethal doses of Cry1Ab Bt toxin orexploited plant tissues that do not express the toxin,and they further implicated that Event 176 hybrids donot satisfy requirements for high dose that arerecommended for resistance management purposes.Pink bollworm, Pectinophora gossypiellaField collected pink bollworm quickly evolvedresistance to Cry1Ac under laboratory selection (Patinet al. 1999, Simmons et al. 1998, Tabashnik et al.2000). Pectinophora gossypiella selected with Cry1Acprotoxin developed 300-fold resistance to Cry1Acprotoxin, and high levels of cross-resistance to Cry1Aaand Cry1Ab protoxin, and low levels of resistance forCry1Bb protoxin (Tabashnik et al. 2000a). Threeselections with Cry1Ac in artificial diet increasedresistance of pink bollworm to >100-fold relative to asusceptible strain (Liu et al. 2001).Tobacco caterpillar, Spodoptera spp.In general, Spodoptera spp. larvae are not verysusceptible to the Cry toxins (Strizhov et al. 1996).However, Cry1C toxin had been reported to be toxicagainst S. exigua (Visser et al .1988) and Spodopteralittoralis (Van Rie et al. 1990a). Selection to Cry1Cacaused 850-fold resistance to Cry1Ca and crossresistanceto Cry1Ab, Cry9C, and Cry2A, as well as toa recombinant Cry1E-Cry1C fusion protein in S.exigua (Moar et al. 1995), while in S. littoralis, 500-fold resistance to Cry1Ca and partial cross-resistance toCry1D, Cry1E, and Cry1Ab has been recorded(Muller-Cohn et al. 1996).BASIS for DEVELOPMENT of RESISTANCE Mutations ininsects that cause disruption of any of the stepsinvolved in the mode of action could confer resistanceto Bt (Heckel 1994). Decreased solubilization of the Btcrystal, decreased cleavage of the full-length Bt proteininto an active fragment, increased proteolytic digestionof the active fragment, decreased binding of the activefragment to the midgut epithelium, and decreasedfunctional pore formation are the major changes in theBt toxicity pathway responsible for evolution ofresistance (Gill et al. 1992). Previous genetic andbiochemical analyses of insect strains with resistance toBt toxins has indicated that: (i) resistance is restrictedto single group of related Bt toxins, (ii) decreased toxinsensitivity is associated with changes in Bt-toxinbinding to sites in brush-border membrane vesicles ofthe larval midgut, and (iii) resistance is inherited as apartially or fully recessive trait. If these threecharacteristics are common to all resistant insects,specific crop-variety deployment strategies couldsignificantly diminish problems associated withresistance in field populations of the target pests(Gould et al. 1992). Recent studies have shown that thegenetic basis of resistance to Bt toxins in insects issimilar to resistance to chemical insecticides, which isconferred by multiple physiological mechanisms underindependent genetic control. In Heliothis, the existenceof separate, independently assorting resistance geneshas already been confirmed by linkage analysis withmarker loci (Heckel 1994). Heckel et al. (1997)identified a major Bt- resistant locus in a strain of H.virescens exhibiting up to 10000-fold resistance toCry1Ac toxin. Despite many potential mechanisms ofresistance, the best-characterized and most widelyobserved mechanism of resistance to B. thuringiensis isreduced binding of toxin to midgut membranes (VanRie et al. 1990b). Changes in the binding affinities oftoxin receptors on the brush border membranes of theinsect midgut have been identified in Bt resistant P.interpunctella (Van Rie et al. 1990b), P. xylostella(Ferre et al. 1991), H. virescens (MacIntosh et al.1991), and Trichoplusia ni (Ballester et al. 1994).Cry1Ab and Cry1Ac have the same receptor in themidgut of O. nubilalis, with the receptor having ahigher affinity for Cry1Ab than for Cry1Ac (Denolf etal. 1993).Studies on a field population of P. xylostella havealso suggested that, apart from reduced binding, otherbiochemical mechanisms are involved in resistance toBt (Martinez-Ramirez et al. 1995). Some evidence forreduced conversion of protoxin to toxin and increaseddegradation of toxin also has been reported (Forcada etal. 1996, Oppert et al. 1994, 1997). In H. armigera, theexcessive degradation of protoxin in midgut juicetriggered by receptor binding of activated toxin waspresumed to be responsible for low sensitivity of theinsect to Bt (Shao et al. 1998). Toxin binding inresistant T. ni selected with Cry1Ab did not correlatewith resistance, since there was no cross-resistance to18

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Cry1Ac (Estada & Ferre 1994), which shares the samebinding site of Cry1Ab as demonstrated in O. nubilalismidgut membrane (Denolf et al. 1993).When the midgut proteinases from resistant strainof European corn borer were characterized, there was a35% decreased hydrolyzing efficiency in activation ofBt protoxin compared with the susceptible strain(Huang et al. 1999). However, in studies by Liu et al.(2000), Cry1C toxin was found to be significantly moretoxic than was Cry1C protoxin to resistant strain ofdiamond back moth, but not to susceptible strain. Ifreduced conversion of Cry1C protoxin to toxin is thesole mechanism of resistance, both susceptible andresistant larvae should be equally susceptible to Cry1Ctoxin. Further, they observed similar binding of 125I-Cry1C to brush border membrane vesicles from theCry1C resistant and susceptible strains and concludedthat reduced binding of Cry1C to midgut target siteswas not a mechanism of resistance in diamondbackmoth. Mohan & Gujar (2003) also found no differencesin proteolytic patterns of Cry1A protoxins in bothsusceptible and resistant populations of diamondbackmoth. They also stated that the differences insusceptibility of two populations to B. thuringiensisCry1Ab were not due to midgut proteolytic activity.McGaughey et al (1998b) indicated that apart fromtoxin solubility and/or proteinase activation in theinsect midgut, postbinding events such as receptoraggregation, pore formation, ionic fluxes, and insectrecovery may also be involved in resistancedevelopment. Following Cry1Ac ingestion by H.virescens, similar histopathological changes wereobserved in midgut epithelium in both susceptible andresistant colony (Forcada et al. 1999, Martinez-Ramirez et al. 1999), suggesting that resistance is dueto a more efficient repair (or replacement) of damagedmidgut cells (Ferre & VanRie 2002).Research conducted over the past 10 years hasindicated that it is likely that the increased use of Bttoxins from transgenics will result in a rapid evolutionof resistance in insects (Gelernter 1997). However,selection of plants for horizontal resistance is moredurable rather than vertical resistance, and the currentresearch on transgenic plants, particularlyincorporation of the Bt delta endotoxins into crops forcontrol of insects appears to be proceeding on a verticalresistance model, based on complete resistanceconferred by one or a few genes. These varieties, likethose produced through conventional resistancebreeding, may become susceptible to the target pests.This may undervalue the benefits of Bt in IPMapproaches (Waage 1996), as it runs the risk ofbreakdown of resistance in the long-term.It may be uneconomic to develop Bt-transformedcrops unless we develop strategies to extend theirusefulness. Wigley et al. (1994) proposed a plan inwhich the major elements to be considered fordeploying Bt genes among crops are: (i) assess the riskof Bt resistant insects evolving and dispersing out ofthe crop to infest others; (ii) characterize the diversityof Bt protein binding sites in the guts of keypolyphagous pests; and (iii) use the above informationto deploy Bt genes among different transgenic crops.RESISTANCE MANAGEMENT Resistant managementstrategies require detailed knowledge of the toxins'mode of action and genetic response of resistantinsects. Unfortunately, insects show great variability intheir genetic responses to Bt toxins. Schnepf et al.(1998) emphasized that laboratory selectionexperiments may give rise to very different outcomesfrom field situations. However, several resistancemanagement strategies have been proposed to delayadaptation to Bt-transgenic crops by pest populations(McGaughey & Whalon 1992, Raymond et al. 1991,Tabashnik 1994). The most promising with currentlyavailable technology is the use of refuges of nontransgeniccrops, augmented wherever possible, withhigh toxin expression in the plants and avoidingmosaics of different toxins and pesticides (Roush1997a).THE REFUGE STRATEGY The primary strategy fordelaying insect resistance to transgenic crops underlarge monocultures is to provide refuges of non-Bt cropplants that serve to maintain Bt-susceptible insects inthe population. This potentially delays the developmentof insect resistance to Bt crops by providingsusceptible insects for mating with resistant insects (Liu et al. 1999).The refuge strategy is expected to work ifresistance to Bt is inherited as a recessive trait. Thebasic goals of the mixture strategy are two fold: (i)reduce the difference in fitness between susceptible andresistant insects, and (ii) reduce the degree to which aresistant insect can pass on its phenotypic trait to itsoffspring. Refuges can consist of fields planted withnon-Bt plants or of non-Bt plants within the Bt plants.The large numbers of susceptible insects that surviveon the refuge plants are then available to mate with thesmall number of resistant insects that survive on the Btplants. The offspring of susceptible (SS) x resistant(RR) matings will be RS, and therefore, will notsurvive when they feed on high dose Bt plants.The US Environmental Protection Agency (EPA),which regulates transgenic pesticidal crops, believesthat scientifically sound long-term insect resistancemanagement (IRM) strategies are essential to theprotection of Bt microbial pesticides, transgenics, andreduction in the risks from the use of pesticides. TheEPA has imposed mandatory IRM requirements for Btcotton. Two structured refuge requirements have beenimposed: 4% unsprayed or 20% sprayed crops (Matten2000), and the refuge fields must be within 0.8 km oftheir Bt fields (EPA/ USDA 1999). Obviously,19

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2enforcing a similar system for small holding farmerswill not be possible in most parts of Asia. In a typicalvillage in Asia, it is unlikely that all farmers will plantBt crops on all their land, and farmers grow severaldiverse crops, which serve as hosts for H. armigera. Insuch a scenario, it may not be necessary to enforce thecultivation of refuge crops (Sharma and Ortiz 2001). Btgenes will be one of many factors that the farmers willconsider when choosing which varieties to grow. Thegovernments can promote the maintenance of refugesby restricting the number and diversity of Bt cultivarsthat can be released. For example, in the Indian state ofPunjab, rice farmers grow traditional Basmati varietiesand modern semi-dwarf varieties. Stem borer damageis higher in basmati varieties, and thus the governmentcould authorize the release of Bt-transformed basmativarieties, but not Bt-transformed semi-dwarf varieties(Cohen 2000).Although Bt cotton that produces Cry1Ac toxinhas been effective against pink bollworm (Patin et al.1999, Tabashnik et al. 2000b), the slower developmentof resistant larvae on Bt cotton as compared tosusceptible larvae on non-Bt cotton could reduce theprobability of mating between susceptible and resistantinsects, and this asynchrony could reduce the expectedbenefits of the refuge strategy (Liu et al. 1999, Liu etal. 2001, Storer et al. 2001). Though there was slowlarval growth, the corn borer larvae were successful incompleting development on transgenic corn plants,causing similar amounts of damage as on non-Bt plants(Storer et al. 2001). Each insect/Bt crop system mayhave unique management requirements because of thebiology of the insect, but the studies have validated theneed for a refuge (Shelton et al. 2000). Therefore, caremust be taken to ensure that refuges, particularly thosesprayed with insecticides, produce adequate numbersof susceptible insects. Models and experimental datashowed that separate but adjacent refuges might besuperior to other strategies for insects that can movebetween plants in their larval stage (Shelton et al.2002).A concern is often raised that insect damage innon-Bt fields will increase after introducing Bt crops.The implication is that farmers will be even less likelyto grow non-Bt crops because of the increased damage,and therefore there will be even fewer refuge fields.However, Cohen (2000) suggested that withdiamondback moth on Bt collards and the Europeancorn borer on Bt maize, many of the moths that emergefrom fields of non-Bt crops would disperse and laytheir eggs in Bt fields. In contrast, very few moths willemerge from Bt fields and move from Bt fields to non-Bt fields. As a consequence, insect damage in non-Btfields may decrease if most fields are planted with Btcrops.There is also a debate regarding the spatial designof the refuge system (separate/seed-mixture) to beadapted. Roush (1997a) pointed out that seed mixescan actually promote resistance development forinsects that move from plant to plant. There is noevidence to show that moths can detect whether or nota plant contains Bt toxin. In some studies, it has beenfound that after the feeding begins, caterpillars moveaway from Bt plants faster than from non-Bt plants, butvery few larvae crawl far enough to move from onefield to another. Ramachandran et al. (1998) found thatP. xylostella larvae move away from transgenic canolaplants within 24 hours. Similarly, H. virescens and H.zea larvae are known to move between plants, so seedmixtures might not work. For endophytic insects suchas P. gossypiella and other stem and root-feedingspecies with limited larval and adult movement,within-field refuge would be best (Gould 1998). Mallet& Porter (1992) pointed out that in seed mixture refugesystem, if the pest's feeding stages could move betweenplants, instead of ingesting a high dose of toxin or notoxin at all, they would often consume intermediatedoses nullifying the advantages of high-dose refuge.The same argument can be extended to transgenicplants with tissue specific expression of toxins.Because of the importance of maintaining appropriaterefuges, insect biology and behavior should also to beconsidered for implementing a refuge system that ispractical and economic.Increasing the size of the refuge delays thedevelopment of resistance. Some workers have calledfor refuges as large as 50%, if farmers are allowed tospray them, which may present a dilemma and reducefarm profitability (Gould & Tabashnik 1998). On theother hand, farmers may be reluctant to sacrifice alarge number of refuge plants to insects just to maintainsusceptible alleles. In China, H. armigera naturallypossesses a vast refuge as it can feed on corn, soybean,peanut, and many other crops. Studies that havemonitored the sensitivity of H. armigera fieldpopulations to Bt insecticidal protein Cry1Ac from1998 to 2000 indicated that H. armigera is stillsusceptible to Cry1Ac protein (Wu et al. 2002b).Although development of H. armigera on Bt cottonwas much slower than on common cotton, there was ahigh probability of mating between populations fromBt cotton and other sources due to scattered emergencepattern of H. armigera adults and overlap of secondand third generations. Thus, in a cotton, soybean, andpeanut mix system, non-cotton crops provided a naturalrefuge (Wu et al. 2002a). As indicated earlier in thediverse cropping systems of the tropics (Sharma et al.2001), where the insects have several alternative andwild hosts, there may not be any need to grow therefuge crops.FUSION GENE STRATEGY Theoretical models suggestthat pyramiding two dissimilar toxin genes in the sameplant has the potential to delay the onset of resistance20

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2much more effectively than single-toxin plants releasedspatially or temporally, and may require smallerrefuges (Roush 1997b). Because of diversity among Bttoxins found in nature, one of the most temptingresistance management strategies is to use two or moreof these toxins in mixtures, rotations, or sequences.Laboratory as well as field studies have beenconducted to evaluate the efficacy of dual proteintransgenic crop plants against several lepidopteranpests (Greenplate et al. 2000a, Stewart & Knighten2000, Stewart et al. 2001). The basis for this strategy issometimes referred to as "redundant killing" becauseinsects adapted to one toxin may be susceptible to thesecond toxin. If the plants contain two Bt toxins at ahigh dose, insects that are able to survive on a plantwith one high-dose toxin are rare, and insects that areable to survive on plants with two high-dose toxins willbe very rare. If such insects are homozygous forresistance alleles for two different genes, and if thefrequency of the allele for resistance to each gene is10-3, then insects of the genotype R1R1R2R2 willoccur at a frequency of only 10^-12, i.e., 1 out of 1trillion. Because such insects will be very rare, fewersusceptible insects will be needed to ensure thatresistant insects do not mate with each other.Therefore, fewer refuge fields will be necessary,although it is still very important to have some refugefields.Similar levels of Cry1Ac have been reported innear isogenic lines of cotton expressing either Cry1Acalone or Cry1Ac and Cry2Ab (Greenplate et al.2000b). Activity of single and double toxin genotypesremained greater than the conventional cottons againsttobacco budworm. However, Bollgard II, with doubletoxin, may have greater efficacy against lepidopterathat mainly feed on reproductive structures. Increasedactivity of Bollgard II (Cry1Ac and Cry2Ab) may bedue to increased potency of Cry2Ab, increased overallexpression level of Cry2Ab, or possibly a synergisticcombination of Cry1Ac and Cry2Ab.(Adamczyk et al.2001). Dual toxin (Cry1Ac and Cry2 Ac) Bt cottonswill provide substantially better control of H. zea, S.frugiperda, and S. exigua compared with the existingsingle toxin (Cry1Ac) Bt cultivars, and may not requiresupplemental insecticidal applications (Stewart et al.2001). Hybrid rice plants expressing a fusion gene,Cry1Ab and Cry1Ac, under the influence of rice actin1promoter are highly resistant to the larvae of bothleaffolder and yellow stem borer (Tu et al. 2000). Theexpression level of the fusion gene (20 ng-1mg solubleprotein) in the genome was sufficient to control thelepidopteran insects (the LD 50 for yellow stem borerneonate is 7.58 mg-1ml diet, whereas that for stripedstemborer is 7.41 mg-1ml diet) (Attotham et al. 1994).Serine protease inhibitors synergized Bt againstfour species of moths and Leptinotarsa decemlineata(MacIntosh et al. 1990). Lee et al. (1996) found that acombination of Cry1Ac and Cry1Aa exerted asynergistic effect on gypsy moth larvae, whereas acombination of Cry1Aa and Cry1Ab was antagonistic.Hence, while considering a pyramiding approach, anexamination of whether co-expression of multiple toxingenes will have a synergistic effect needs to beundertaken. Similarly, if Bt toxin genes are to beintegrated with protease inhibitor genes, proteaseinhibitors that do not affect the protease-mediatedcleavage to release activated Bt toxin but that are stillcapable of inhibiting digestive process of the insectneed to be engineered.The strategy of "pyramiding," i.e., combining twotoxins in a single transgenic plant will, at best,substantially reduce the size of the needed refuge andat worst, produce resistance to both toxins in the sameamount of time as for a single toxin (Roush 1997b).Cross-resistance among toxins and the ability of insectsto develop resistance to multiple toxins will limit thesuccess of this approach (Roush 1998). Studies haveshown that there are large differences in the crossresistancespectrum of the insect species that have beenselected for resistance using single toxins or toxinmixtures. Polygenic inheritance and the existence ofmultiple mechanisms of resistance may be involved inbroad-spectrum resistance, and may limit the use ofmultiple toxin strategies for managing resistance(McGaughey 1994). Although, the independence ofCry1C resistance from Cry1A resistance indiamondback moth suggests that Cry1C and Cry1Atoxins might be useful in rotations or mixtures fordelaying resistance (Liu & Tabashnik 1997), thedominance of resistance can vary for a given pest fromdifferent locations. However, pyramiding of two ormore insecticidal genes in the same plant is apromising long-term strategy for delaying resistance,and one which is more forgiving on refuge size. Theso-called, high dose strategy, combined with the use ofrefuges, is widely agreed to be the best technicalapproach for managing resistance, and evidence isaccumulating that 'separate' refuges are more effectiveat conserving pest susceptibility than 'mixed' refuges(Cannon 2000).THE HIGH-DOSE APPROACH Doses of toxins that do notmake life hard for susceptible individuals, either bykilling them or by reducing their reproductive output,do not select for resistance. On the other hand, dosesthat are sufficient to kill all individuals in a population,including the most resistant genotypes, do not select forresistance either, because no one is favored bydiscrimination. However, slow decay of toxin residuesmeans that there will almost certainly be a time periodwhere discrimination works strongly in favor ofresistant individuals in a population (Tabashnik &Croft 1982). Low-dose insecticide applications havebeen shown to create high risks of resistance21

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2development (Georghiou & Taylor 1977) and thetheoretical potential for spraying crops with extremelyhigh doses of one or more insecticides has beendiscussed often (Roush 1989, Tabashnik & Croft1982). The "high-dose refuge" strategy is the mostwidely used and has been implemented in NorthAmerica (Alstad & Andow 1995). When an insecticidespray kills 95% of the susceptible (SS) individuals, thesurvival of RS individuals is likely to be significantlyhigher, unless the alleles governing resistance happento be phenotypically recessive (i.e, the RS and SSinsects are physiologically identical). Instead of hopingthat resistance is phenotypically recessive, the highdose approach attempts to make resistance alleles"effectively recessive" even if they are notphenotypically recessive (Gould 1998). Similarly, dosethat is insufficient to kill the insects bearing one copyof a major resistance allele renders resistancefunctionally partially dominant. Hence, the onlycommercially available approach to reduce thelikelihood of resistance development is the use of ahigh dose of a single gene, producing 25 times thetoxin concentration needed to kill susceptible insects incombination with a refuge.High concentrations of Cry1Ac in bolls oftransgenic cotton are essential for achievingfunctionally recessive inheritance of resistance (Liu etal. 2001). Further, extensive planting of transgenic cornhybrids having sub-optimal production of the toxin andresulting in only moderate effects on H. zea wouldraise concerns about the rapid evolution of resistance(Storer et al. 2001). If transgenic plants could be madeto express enough toxins to overcome all homozygousresistance alleles, the crop in question would become anon-host. The lack of a "high dose" in current Bt cottoncultivars for H. armigera and the small scaleproduction systems of cotton indicates that the "highdose/refuge" resistance management strategy is notfeasible for Bt cotton in northern China (Zhao et al.2000a). Under these circumstances, supplementalcontrol of H. armigera with insecticides is essential togrow Bt cotton for a longer period (Ru et al. 2002).Resistance in insects to Bt can be dramatically reducedthrough the genetic engineering of chloroplasts inplants. Several copies of the Bt genes could beexpressed per cell via the chloroplast genome asopposed to only two copies via the nuclear genome in adiploid cell. The Cry2Aa2 protoxin levels inchloroplast-transformed tobacco leaves are between 2to 3% of total soluble protein, and are 20-to-30-foldhigher than current commercial transgenic plants (Kotaet al. 1999). If a toxin is consistently produced by aplant at a highly toxic concentration without having anegative effect on yield, and the toxin does not affectnon-target organisms, then the constraints on high dosestrategy would be quite low.Another serious concern regarding the success ofhigh dose strategy is that the hypothesis of resistancebeing recessive does not hold in different insectspecies. Inheritance of resistance showed incompletedominance in O. nubilalis to a commercial preparationof Bt (Huang et al. 1999), and in H. virescens toCry1Ab (Sims & Stone 1991). While, Tabashnik etal.(1998) demonstrated dominant resistance to Cry1Aain a strain of P. xylostella having field-evolved Btresistance.CONTROLLED EXPRESSION of TOXINS Mono-cultivationof Bt transgenic crops is likely to select intensely forresistance because pests will be exposed to Bt evenwhen they are not causing economic damage (Mallet &Porter 1992). The degree of yield reduction caused by apest population is dependent on its density, as well ason when and where insects feed on the plants.Expression of toxin coding genes could be limited tovulnerable plant parts, and at times when toxicity isneeded most. If a pest causes no damage when it feedson mature leaves, but causes severe stunting when itfeeds on buds and developing leaves, then toxinproduction only in buds would be useful. Having Btexpressed in plants so that the insect population issubjected to selection pressure for particular periods oftime (e.g., through an inducible promoter) or inparticular plant parts (e.g., through tissue-specificpromoters) may provide larger refuges for susceptiblealleles both within the field and within a region whileat the same time minimizing the crop loss (Roush1997b). This can be achieved by using gene constructshaving a tissue specific promoter.In P. xylostella, resistance to Bt declined whenexposure to insecticide ceased (mean R = -0.19). Infour other pests (H. virescens, L. decemlineata, Muscadomestica and P. interpunctella), resistance to Btdeclined slowly or not at all (mean R = -0.02) in theabsence of exposure to Bt (Tabashnik et al. 1994 ).Similar loss of resistance in O. nubilalis was observedin the absence of selection pressure (Bolin et al. 1999).This can be exploited for formulating resistancemanagement strategies by enforcing completerestriction on cultivation of certain Bt cultivars for aspecified period.Solutions to resistance management involvecomplex strategies. The track record of resistancemanagement for chemical pesticides is notencouraging. The wisdom gained from previouspesticide failures should provide impetus for theproactive development and implementation ofmanagement strategies for transgenic crops. Keepingthis in view, Cohen (2000) made four practicalrecommendations for promoting the sustainable use ofBt crops, based on existing knowledge of the principlesof resistance management:22

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Do not release Bt varieties that do not have a highdose of toxin. Toxin titers of 2 µg/g of leaf freshweight or 0.2 % of soluble leaf protein have beenshown to act as high doses against most insectpests of crops.Release only Bt cultivars that have two Bt toxingenes, which are not closely related to each other,and both should be expressed at a high dose.Do not release Bt-transformed versions of allpopular crop varieties. Some popular non-Btvarieties should remain available to improvechances that some non-Bt fields (refuges) willexist.Implement resistance monitoring programs toserve as an early warning system for governmentsand farmers and provide valuable information forimproved deployment of future pest-resistantcultivars.However, the farm-level implementation ofresistance management will face practical and socialobstacles. A survey conducted by US maize growershas shown that in the year 2000, almost 30% of thefarmers failed to comply with the refuge protocolsdesigned to prevent or delay the emergence of insectsresistant to Bt toxins (Dove 2001). Ensuring effectiveresistance management practices is a challenge thatwill require coordination from all sectors (public andprivate) concerned with crop protection, and willrequire the commitment of growers and advisers thatcurrent technology for crop protection is a preciousresource vital to profitable production. There is acontinuing need for interaction between ecologists,geneticists, and plant breeders in determining systemwideimpacts and devising optimal ways of deployinginsect-resistant crops. 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Field performance of transgenic elite commercialhybrid rice expressing Bacillus thuringiensis d-endotoxin. Nat.Biotechnol. 18: 1101-1104.Van Rie, J., Jansens, S., Hofte, H., Degheele, D. & Van Mellart, H.1990a. Receptors on the brush border membrane of the insectmidgut as determinants of the specificity of Bacillus thuringiensisdelta-endotoxins. Appl. Environ. Microbiol. 56: 1378-1385.Van Rie, J., McGaughey, W. H., Johnson, D. E., Barnett, B. D. &Mellaert, H. 1990b. Mechanism of insect resistance to the microbialinsecticide Bacillus thuringiensis. Science 247: 72-74.Visser, B., Van der Salm, J., Van der Brink, W. & Folkers, G. 1988.Genes from Bacillus thuringiensis entomocidus 60.5 coding forinsect-specific crystal proteins. Mol. Gen. Genet. 212: 219-224.Waage, J. 1996. IPM and Biotechnology. Biotechnology and IntegratedPest Management. (ed. Persley, G.J.) CAB International,Wallingford, UK. pp 37-60.Wigley, P. J., Chilcott, C. N. & Broadwell, A. H. 1994. Conservation ofBacillus thuringiensis efficacy in New Zealand through the planneddeployment of Bt genes in transgenic crops. Biocontrol Sci.Technol. 4 (4): 527-534.Wu, K., Guo, Y. & Gao, S. 2002a. Evaluation of the natural refugefunction for Helicoverpa armigera (Lepidoptera: Noctuidae) withinBacillus thuringensis cotton growing areas in North China. J. Econ.Entomol. 95: 832-837.Wu, K., Guo, Y., Lu, N., Greenplate,J. T.& Deaton, P. 2002b. Resistancemonitering of Helicoverpa armigera (Lepidoptera: Noctuidae) toBacillus thuringensis insecticidal protein in China. J. Econ.Entomol. 95: 826-831.Zhao, J. H., Rui, C. H., Lu, M. G., Fan, X.. L. & Ru, L.J. 2000a.Monitoring and management of Helicoverpa armigera resistance totransgenic Bt cotton in North China. Resist. Pest Manage. 11(1):28-31.Zhao, J. Z., Collins, H. L., Tang, J. D., Cao, J., Earle, E.D., Roush, R. T.,Herrero, S., Escriche, B., Ferre, J. & Shelton, A. M. 2000b.Development and charecterization of diamondback moth resistanceto transgenic broccoli expressing high levels of Cry1C. Appl.Environ. Microbiol. 66: 3784-3789.Zhao, J. Z., Zhao, K. J., Lu, M. G., Fan, X. L. & Guo, S. D. 1998.Interactions between Helicoverpa armigera and transgenic Btcotton in North China. Sci. Agricul. Sini. 31: 1-6.Zoerb, A.C., Spencer, T., Richard, L. H., Robert, J. W. & Siegfried, B. D.2003. Larval distribution and survival of second generationEuropean corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera:Crambidae) on Event 176 Bt corn. Crop Protection 22: 179-184.S. V. S. GopalaswamyInternational Crop Research Institute for Semi-Arid TropicsHyderabad, Andhra Pradesh.India& Acharya N G Ranga Agricultural University25

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Hyderabad, Andhra PradeshIndiaG. V. SubbaratnamAcharya N G Ranga Agricultural UniversityHyderabad, Andhra PradeshIndiaH. C. SharmaInternational Crop Research Institute for Semi-Arid TropicsHyderabad, Andhra PradeshIndiaGenerating Baseline Data for Insecticide Resistance Monitoring in Cotton Aphid, Aphis gossypii GloverABSTRACT The baseline susceptibility data weregenerated for the six commonly used insecticides viz.,thiamethoxam, imidacloprid, dimethoate, methyldemeton, acephate, and monocrotophos in cottonecosystems for the field population of Aphis gossypii.Populations were collected from the cotton fields of theDepartment of Cotton, Agricultural College andResearch Institute, TNAU, Coimbatore, India. IRACmethod No. 8 developed and recommended byInsecticide Resistance Action Committee (IRAC) witha slight modification was used for arriving the lethalconcentrations. The base line susceptibility data werecreated for seven generations. The LC50 values variedfrom 0.3412 to 1.0414 for thiamethoxam, 0.4583 to1.8055 for imidacloprid, 3.0096 to 10.6924 fordimethoate, 12.598 to 49.2606 for methyl demeton,1.4615 to 5.3284 for acephate, and 1.1866 to 3.7057for monocrotophos. The LC95 values varied from10.8617 to 35.2153 for thiamethoxam, 17.9171 to43.4310 for imidacloprid, 49.1667 to 629.6511 fordimethoate, 418.4538 to 1174.6270 for methyldemeton, 36.1800 to 130.4890 for acephate, and24.9571 to 139.4943 for monocrotophos.KEY WORDS: Insecticide resistance, Aphis gossypii,diagnostic dosesINTRODUCTION The importance of Aphis gossypiiGlover as a cotton pest is increasing throughout theworld (Leclant and Deguine, 1994). High aphidpopulations may stunt and retard cotton seedlinggrowth and development as a result of its feeding. Lateseason populations can cause decreased fiber quality asthe result of stickiness and the development of sootymould associated with honeydew dropped onto cottonfibers (Isely, 1946). There has been a general decline inthe effectiveness of several insecticides to control A.gossypii. The intensity of aphid infestations hasincreased over the last ten years and the use ofinsecticides to control aphids is questioned.The pest problem is aggravated more rapidly dueto control failures in many areas. Though controlfailure may be due to many factors, one of the majorfactors is the development of resistance to insecticides.The chief objective in resistance monitoring is toexaggerate the differences between susceptible andresistant individuals such that the frequency ofmisclassification is greatly reduced (Ffrench-constantand Roush, 1990). This is fulfilled by fixing thediagnostic doses.Resistance to A. gossypii is in the initial stages ofdevelopment and no systematic work has been done sofar on monitoring of insecticide resistance in India as ithas been done in Amrasca devastans (Distant) (JayaPradeepa and Regupathy, 2002), Helicoverpa armigera(Hub), Plutella xylostella (Linn.), and Spodopteralitura (Niranjan Kumar and Regupathy, 2001). Giventhe background, the present study was undertaken todetermine the diagnostic doses for the commonly usedinsecticides in cotton for A. gossypii.MATERIALS and METHODS The test insects werecollected from the cotton field, Department of Cotton,Agricultural College and Research Institute, TNAU,Coimbatore, India. The population was maintained forseven continuous generations without exposure topesticides under the laboratory conditions forgenerating the baseline data, i.e. fixing diagnosticdoses.The dilutions required were prepared from thecommercial formulations of insecticides using distilledwater. The dosages were attained after preliminaryrange finding studies for constructing logconcentration-probit-mortality(lcpm) lines (Regupathyand Dhamu, 2001).The wingless adults aphids of ca 1.45mm size andweighing ca 0.19mg were taken from the culturemaintained for the treatment. Each replicationconsisted of 10 aphids and there were threereplications. Bioassays were conducted following theprocedure based on the standard Bemesia tabaciGennadius susceptibility test, IRAC method No.8developed and recommended by the InsecticideResistance Action Committee, with slight modification.The experimental setup consisted of twodisposable cups, one as an inner test chamber and theother as an outer water reservoir. The cup that servedas the inner test chamber was taken and a hole waspierced in the centre of the bottom side of the cup.The young green uncontaminated leaves wereselected and the petiole was cut to a length ofapproximately four cm. The leaves were dipped in theconcentrations for five seconds holding the leaf by thepetiole with fine forceps. Care was taken to avoid thedamage to the petiole. Then the leaves were left for26

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2drying in the open air by placing the leaves on the filterpaper (approximately five minutes). The petiole of theleaf was passed through the inner cup and the winglessaphids were released into the inner cup at the rate of 10aphids per cup and the cup was covered with muslincloth tightened with rubber band. A small amount ofwater was placed in a second cup and the test cup wasplaced inside that, so that it was supported by theprotruding petiole. Observations on mortality of aphidswere recorded after 48 h. The results were expressed aspercentage mortality.RESULTS and DISCUSSION The LC50 values varied from0.3412 to 1.0414 for thiamethoxam, 0.4583 to 1.8055for imidacloprid, 3.0096 to 10.6924 for dimethoate,12.598 to 49.2606 for methyl demeton, 1.4615 to5.3284 for acephate, and 1.1866 to 3.7057 formonocrotophos. Thiamethoxam was the most toxicpesticide. The acute toxicity of other insecticides basedon LC50 was in the order of imidacloprid >monocrotophos > acephate > dimethoate > methyldemeton for all the seven generations tested.The LC95 values varied from 10.8617 to 35.2153for thiamethoxam, 17.9171 to 43.4310 forimidacloprid, 49.1667 to 629.6511 for dimethoate,418.4538 to 1174.6270 for methyl demeton, 36.1800 to130.4890 for acephate, and 24.9571 to 139.4943 formonocrotophos. The acute toxicity was in the order ofthiamethoxam > imidacloprid > acephate >monocrotophos > dimethoate > methyl demeton for F1and F2 generations, and thiamethoxam > imidacloprid> monocrotophos > acephate > dimethoate > methyldemeton for rest of the generations tested.The susceptibility was gradually increased with thesucceeding generation, which is evident from thedecline in LC50 and LC95 values to all the insecticidestested. The extent of increase was greater for methyldemeton and dimethoate respectively. Thesusceptibility baseline data are not generated so far forthese insecticides taken up for the study. Hence, theLC95s of the insecticides were considered asdiscriminating doses for monitoring the fieldpopulations for their resistance to these insecticides.From the acute toxicity studies conducted in ourlaboratory, the discriminating doses (ppm) fixed were10 for thiamethoxam, 20 for imidacloprid, 50 fordimethoate, 400 for methyl demeton, 40 for acephate,and 20 for monocrotophos.Based on the slope function and increasedsusceptibility, the discriminating dose screen was fixedfor monitoring the level of insecticide resistance infuture.REFERENCESFfrench-constant, R.H. and R.T. Rousch. 1990. Resistance detection anddocumentation; the relative role of pesticidal and bio chemicalassays. In: Pesticide resistance in arthropods. (Eds.) R.J.Rousch andB.E.Tabashnik, Chapman and Hall; Newyork and London, pp 4-38.Isely, D. 1946. The cotton aphid. Arkansas Agric. Exp. Stn. Bull. 464.Jeya Pradeepa, S. and A. Regupathy. 2002. Generating baseline data forInsecticide Resistance Monitoring in cotton Leafhopper, Amrascadecastans (Distant). Resistant Pest Management Newsletter, 11(2).Leclant, F. and J.P. Deguine. 1994. Cotton aphids. In: Insect pests ofcotton. (Eds) G.A.Mathews and J.P.Tunstall. C.A.B.: 285353.Niranjankumar, B.V. and A. Regupathy. 2001. Status of insecticideresistance in tobacco caterpillar Spodoptera litura (Fabricius) inTamil Nadu. Pestic. Res. J. 13 (1): 86-89.Regupathy, A. and K.P.Dhamu. 2001. Statistics work book for InsecticideToxicology. Softech Computers, 206p.Praveen P. M. and A. RegupathyDepartment of Agricultural EntomologyAgricultural College and Research InstituteTamil Nadu Agricultural UniversityCoimbatore-641 003IndiaBaseline Susceptibility and Quantification of Resistance in Plutella xylostella (L.) to SpinosadABSTRACT Baseline susceptibility to spinosad in a P.xylostella population was determined by topicalbioassay. Significant variations in the LC50 valuesranged from 0.000250 in the 5th generation to0.000299 in the 1st generation. The resistance ratio forthe 7th generation as compared to the 1st generation27

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2was 0.90, which indicated that resistance to spinosaddid not develop in P. xylostella after continuousselection at least up to the 7th generation. Crossresistanceto commonly used insecticides was alsostudied. The toxicity of commonly used insecticideswas also calculated against spinosad-selected P.xylostella. Comparisons of LC50 values indicated thatmonocrotophos (1.21278%) was the least toxic of allthe insecticides whereas cypermethrin (0.0276%) wasfound to be the most toxic. The relative toxicity ofcypermethrin, dichlorvos, malathion, endosulfan, andcarbaryl reveal that these insecticides were 43.94,20.80, 5.85, 4.24, and 4.17 times more toxic thanmonocrotophos.INTRODUCTION The diamondback moth (DBM),Plutella xylostella L. (Lepidoptera: Plutellidae) is oneof the major constraints in the profitable cultivation ofcole crops. The pest occurs in endemic form with highpopulation densities on early and late sowncauliflower. In case of severe infestation, the growinghearts are also damaged, affecting the production ofmarketable curds. The control of P. xylostella hasdepended primarily and extensively on the use ofinsecticides recommended for the last over forty years(Syed, 1992). However, the promiscuous use of anumber of commercial insecticides lead to thedevelopment of resistance in this pest in most countriesof Southeast Asia (Georghiou, 1981). Many factors likepronounced cultivation of early and late varieties ofcauliflower, intensive use of conventional insecticides,and prospects of the higher value of the crop duringoff-season have been outlined for its extraordinarypropensity to develop resistance to all classes ofcompounds. By and large one of the countermeasuressuggested to combat the menace of resistance is theintroduction of, and switching over to, newer,ecofriendly, and more potent insecticides.Spinosad is a new class of polyketide-derivedmacrolide effective against a broad range of pestsbelonging to orders lepidoptera, diptera, andhymenoptera (Sparks et al., 2001). It contains a mixtureof two very active principles: Spinosyn A andSpinosyn D, and is derived from a new species of soilbacterium Saccharopolyspora spinosa which acts bothas a contact and a stomach poison.Electrophysiological evidences have demonstrated thatit alters nicotinic currents in neuronal cell bodies andalso disrupts the functions of GABA receptors of smallneurons in the central nervous system (Salgado et al.,1997).Keeping in view the introduction of this chemicalin India and the potential of P. xylostella to developresistance to all classes of compounds, a need wasrealized to generate base line susceptibility data andquantify resistance to spinosad as well as crossresistancewith other chemicals with the aim to detectshifts in susceptibility following its commercial useand to formulate future strategies to manage this pest.MATERIALS and METHODSInsect Rearing: The culture of P. xylostella wasinitiated by collecting about 200 larvae from farmers'cauliflower fields and brought to the laboratory forfurther multiplication at a regularly maintainedtemperature of 24±20C. The larvae were reared oncauliflower leaves till pupation. The adult moths wereallowed to lay eggs in oven-dried glass jars havingcauliflower leaves to serve as substrate for oviposition.They were provided with 10% honey solution fortifiedwith multivitamins for feeding on a cotton swab. Theneonates were provided with fresh cauliflower leavesto feed upon. At every successive instar, the larvaewere shifted to clean jars containing fresh leaves. Thewhole stock was divided into two lots. One lot wasnamed as parental stock and the other was used forexposure to spinosad.Preparation of Insecticidal Concentration: Theproprietary products of all the insecticides were used toprepare one percent stock solution in acetone fromwhich further dilutions were prepared subsequently.Bioassay and Laboratory Selection: FAO methodNo.21 (Busvine, 1980) for topical application ofinsecticide to the larvae with slight modification wasfollowed. Instead of directly treating the larvae withoutany substrate, the larvae released on cut discs ofcauliflower leaves of the size of a petri dish (2.5 cmdiameter) were treated to simulate the application ofinsecticide in the field. To facilitate the movement ofthe larvae on both sides of the leaf disc, the leavesbearing slightly thick midribs and veins were selectedfor cutting the leaf discs. Before spraying, ten 3rdinstar larvae were released on the upper side of the leafdisc as one replication. 1mL each of the insecticidalconcentrations were sprayed on each side of the leafdisc with Potter's tower at 5lb/inch^2 pressure. Allthree replications were maintained for eachconcentration. After the treatment the petri dishes wereshifted to BOD at 24±10C with 60-70% relativehumidity. Larval mortality was recorded after every 72hours of the exposure by counting the larvae as deadwhen they did not resume activity after repeatedproddings. The survivals from the experiments,affording around 85 percent mortality, were reared tothe next generation. The progeny of the first survivinglot was termed the F1 generation and the exposures andselections were conducted up to 7 generations. Theparental strain was also maintained through withoutexposure.Statistical Analysis: Data on mortality was subjected toAbbott's formula for correction wherever required28

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2(Abbott, 1925). LC50 values of allinsecticides were determined by Probitanalysis (Finney, 1971).Quantification of Insecticidal Resistance:The degree of development of resistancethrough different generations wasdetermined by working out LC50 values ineach generation and thus computing theresistance ratio by dividing the LC50 valuefor that generation with LC50 value of theF1.Cross-resistance with Other Insecticides:The studies on cross-resistance ofspinosad-selected strains of P. xylostella tocommonly used insecticides viz.monocrotophos, malathion, endosulfandichlorvos, cypermethrin, and carbarylwere also made as per the proceduredescribed.RESULTS and DISCUSSION Selection pressureof spinosad and responses produced indifferent generations has been presented inTable 1. Significant variations in the LC50values of all seven generations indicatethat there was no consistency in thetoxicity of spinosad to P. xylostella larvae.The LC50 in the 2nd generation(0.000299%) decreased slightly comparedto the 1st generation with a furtherdecrease in the 3rd, 4th, and 5thgenerations. Thereafter, in the 6th and 7thgenerations, it increased slightly but nevercame at par with the F1. Thus, thedecrease in LC50 values in the subsequentgenerations showed slightly increasedsusceptibility. The resistance ratios also showed asimilar trend. The resistance ratio for the 7th generationas compared to the 1st generation was 0.90, whichindicated that resistance to spinosad did not develop inP. xylostella after continuous selection at least up to 7generations. From this, it can be construed thatspinosad can be used commercially as an alternative toparticularly those insecticides against which P.xylostella has developed resistance. As per theavailable literature, such studies have not beenundertaken on the development of resistance tospinosad in P. xylostella. However, the bioefficacy ofspinosad to 10 different strains of Pseudoplusiaincludens (Walker) revealed that LC50 values variedfrom 4.19 to 13.46 ppm (Mascarenhas and Boethal,1997). The results of topical bioassays conducted bySparks et al. (1998) with spinosyn A and D againstHeliothis virescens larvae showed LC50 values rangingbetween 1.28 to 2.56 µg/g.The toxicity of commonly used insecticides wasalso calculated against spinosad-selected P. xylostella(Table 2). The comparisons of LC50 values indicatedthat monocrotophos (1.21278%) was the least toxic ofall the insecticides used against this strain whereascypermethrin (0.0276%) was found to be the mosttoxic. Data pertaining to the relative toxicity ofcypermethrin, dichlorvos, malathion, endosulfan, andcarbaryl revealed that these insecticides were 43.94,20.80, 5.85, 4.24, and 4.17 times more toxic thanmonocrotophos. It could be concluded that spinosadselectedstrains of P. xylostella did not show any crossresistance to these insecticides. As per literaturescreened, no such studies have been carried out so faron the quantification of cross resistance to thespinosad-selected strain of P. xylostella. Since themode of action of spinosad is altogether different fromother insecticides, the chances of any cross-resistancein this case seem to be quite dim (Salgado et al.1997).29

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2REFERENCESAbbott, W. S. 1925. A method of computing the effectiveness of aninsecticide. J. Econ. Entomol., 18: 265-267.Busvine, J.R. 1980. Recommended methods for detection andmeasurement of resistance of agricultural pests to pesticides. FAO,Rome. pp. 44-46.Finney, D.J. 1971. Probit analysis. Third Edition. S.Chand & Co.Ltd.,New Delhi.333 p. Georghiou, G.P. 1981. The occurrence ofresistance to Pesticides in arthropods. An index of cases reportedthrough 1980. FAO, Rome. 373 p.Mascarenhas, R.N. and Boethal, D.J. 1997. Responses of field collectedstrains of soybean looper (Lepidoptera: Noctuidae) to selectedinsecticides using an artificial diet overlay bioassay. J.Econ.Entomol., 90: 1117-1124.Salgado, V.L., Watson, G.B. and Sheets, J.B. 1997.Studies on the modeof action of spinosad, the active ingredient in Tracer Insect Control.Proc. Beltwide Cotton Production Conferences. Memphis, TN,USA. pp. 1082-1086.Sparks,T.C., Thompson, G.D., Herbert, A.K., Hertlein, M.B., Larson,L.L., Worden, T.V. and Thibault, S.T.1998. Biological activity ofthe spinosyns, new fermentation derived insect control agents ontobacco bud worm (Lepidoptera: Noctuidae) larvae. J.Econ.Entomol., 91: 1277-1283.Sparks, T.C., Crouse, G. D. and Durst, G. 2001. Natural products asinsecticides: the biology, biochemistry and quantitative structureactivityrelationships of spinosyns and spinosoids. PestManagement Sc. 57: 896-905.R.K.AroraDivision of EntomologyFaculty of Agriculture, SKUAST-JammuUdheywalla Jammu -180 002IndiaBaseline Susceptibility of Diamondback Moth, Plutella xylostella (Linn.), to New InsecticidesABSTRACT Toxicity of three new insecticides (fipronil,indoxacarb, and diafenthiuron) was assessed against3rd instar larvae of diamondback moth (DBM) using aleaf residue technique. LC50 values of theseinsecticides were observed to be very low indicatingthe high toxicity and potential of these compoundsagainst multi-resistant populations of DBM. Baselinesusceptibility data will be useful to monitor theresponse of DBM to these compounds in future forearly detection of resistance.KEY WORDS Diamondback moth, cabbage, newinsecticides, bioassay, insecticide resistance, baselinesusceptibilityand Joia, 1992). The problem is acute in areas wherevegetables are grown extensively in a staggeredmanner almost throughout the year, particularly aroundand near big cities (Joia et al. 1996). In view of thereports of field control failure and the development ofresistance in the pest, it was considered appropriate toundertake studies to assess toxicity of new moleculestowards multi-resistant populations of DBM. Theobjective was to identify potential compounds forinsecticide resistance management of the pest and toestablish baseline susceptibility of DBM to theseinsecticides.MATERIALS and METHODSINTRODUCTION Large scale and indiscriminate use ofinsecticides for the control of insect pests, necessitatedby ever increasing demand for quality food and betterpublic health has resulted in a number of problems.One of the major problems that has arisen out of theabuse of insecticides is the development of resistancein insect pests to pest control chemicals. Diamondbackmoth (DBM), Plutella xylostella (Linn.), is aubiquitous pest wherever crucifers are grown. It is themost destructive and is a regular pest of cabbage andcauliflower. The pest is known for its propensitytowards quick development of resistance (Georghiou,1990) and there have been instances when this pest hasdeveloped resistance to a new molecule within a fewyears of its introduction (Hama, 1989). The pest hasdeveloped resistance to almost all the recommendedinsecticides belonging to major groups in many parts ofthe world (Talekar and Shelton, 1993) and is becomingincreasingly difficult to control. In India, resistance inP. xylostella to different insecticides has been reportedfrom several states like Punjab, Haryana, UttarPradesh, Karnatka, Tamil Naidu, and Andhra Pradesh(Mehrotra and Phokela 2000). In Punjab, DBM hasdeveloped resistance to quinalphos, fenvalerate,cypermethrin, and several other insecticides (ChawlaTest Insects: Pupae and larvae of diamondbackmoth were collected from infested plants in variouscabbage / cauliflower growing areas of Punjab duringthe period from September to October. The insectswere reared in the laboratory on cabbage leavesobtained from unsprayed crops. Third instar larvae ofuniform size and weight from F1or subsequentgenerations were used for bioassay.Insecticides: Commercial formulations of testinsecticides were procured from the manufacturersdirectly. The products were diluted with water to obtaina range of test concentrations, usually 6 to 7 for each ofthe test insecticides.Bioassay Method: The leaf residue technique(Tabashnik et al., 1987) with slight modifications wasused for exposing larvae to test insecticides. Discs (5cm diameter) of cabbage leaves were dipped in testconcentrations for 5 seconds and dried for 1 hour atroom temperature. Distilled water was used as control.After drying, the discs were placed in glass containers.Around thirty 3rd instar larvae were released per twodiscs for each concentration and the mouth of thecontainer was secured with muslin cloth tied with30

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2rubber band. The test larvae were allowed to feed for48 hours on treated discs. Thereafter, larval mortalitywas recorded and LC 50 worked out using a computerprogram based on Probit Analysis (Finney, 1971).RESULTS and DISCUSSIONFipronil: Fipronil, 5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-(trifluorosulfoxide-1,2-pyrazole), is an insecticide of recent introduction. Thisinsecticide was evaluated in the laboratory for itstoxicity towards DBM larvae. Fipronil proved to bevery toxic to test larvae. The LC50 values of fipronilagainst six populations of DBM ranged from 0.0001 to0.0045% (Table 1). The population from Khanna wasthe most susceptible, and that from Amritsar was theleast susceptible to this insecticide. Low LC50 valuesindicate high toxicity of fipronil to multi-resistantpopulations of DBM. Non-exposure of the pestpopulations to this compound and high inherenttoxicity may be the possible reasons for low LC50values. Low LC50 values (3.17 µg/ml) of fipronil havebeen reported in diet bioassay with DBM larvae(Argentine et al., 2002). The compound has beenreported to be effective against DBM in China (Zhao etal. 1995) and Hawaii (Mau and Gusukuma-Minuta,1999). It has also been registered for the pest oncauliflower in Western Australia (Lancaster and Burt,2001).to DBM larvae from four different populations and theLC50 values and other probit parameters are presentedin Table 3. These data show that this insecticide alsopossesses fairly high toxicity to resistant DBMpopulations but it is not as toxic as previouslydescribed fipronil and indoxacarb. The LC50 valuesvaried from 0.0051 to 0.011%. Zhao et al. (1995) havereported diafenthiuron to be effective against DBM inChina. Solang and Sribhuddachart (2002) havereported that in addition to its high toxicity to DBM,the pest did not develop resistance after selection with25 generations.All of these compounds have been introducedrecently and are still at the evaluation stage underIndian conditions. Apart from inherent toxicity of theseinsecticides, non-exposure of DBM populations tothese chemicals is perhaps the other reason for verylow LC50 values. After further testing, theseinsecticides may prove as alternate control measuresfor DBM in problematic areas. However, to fullyrealize their potential and to increase useful life forlong-term use in effective IRM, these and similar newgeneration compounds will have to be used veryjudiciously. There must be some mechanism toIndoxacarb: Indoxacarb, (S)-methyl-7-chloro-2,5-dihydro-2 [[(methoxycarbonyl) [4-(trifluoromethoxy)phenyl] amino] carbonyl]-indeno{1,2-e}{1,3,4}oxadiazine-4a(3H)-carboxylate, is another newinsecticide, which has been specifically introduced forthe control of lepidopterous insect pests. Bioassaystudies with indoxacarb were also conducted againstmulti-resistant populations of the pest from Jagraon,Samrala, Phagwara, and Khanna and the results aregiven in Table 2. This insecticide was found to beextremely toxic and the LC50 values of this compoundranged from 0.00003 to 0.00007%. Very high toxicityof this insecticide against DBM larvae indicates thehigh potential of indoxacarb. Field trials conducted atthe University of Arizona have shown it to give goodcontrol of DBM infesting cabbage (Umeda et al. 2000).It has also been reported to exhibit synergism withgranulosis virus against DBM (Krishnamoorthy 2002).Mau and Gusukuma-Minuta, (2002) have reported itsuse in insecticide resistance management of DBMHawaii.Diafenthiuron: Another introduction of recent times,diafenthiuron, 3-(2,6-diisopropyl-4-phenoxyphenyl)-1-tert-butyl-thiourea, is a new type of thiourea. It is apro-insecticide. After application, it gets converted tocarbodiimide, which is an inhibitor of mitochondrialATPase. This compound was also tested for its toxicity31

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2regulate the use of insecticides by farmers. Educationof farmers and formulation of guidelines and strictcompliance can prove very useful in this direction.Based on bioassays of these compounds withDBM populations from different locations in the state,LC50s of 0.0001%, 0.00003%, and 0.0051%, forfipronil, indoxacarb, and diafenthiuron respectively,have been worked out. The baseline susceptibility datawill be very useful for monitoring of susceptibilitystatus of DBM to these insecticides in the future andmay help in early detection of resistance developmentin the pest to these compounds.ACKNOWLEDGEMENTS The present studies wereconducted under the research scheme "Insecticideresistance management in diamondback moth, Plutellaxylostella (L.) on cole crops." The authors are thankfulto the Indian Council of Agricultural Research, NewDelhi for funding the same. The authors gratefullythank Professor and Head, Department of Entomology,PAU Ludhiana for providing the necessary facilitiesand research associates and other staff for helping tocarry out various experiments during the course of thepresent studies.REFERENCES1 Argentine J.A., Jansson RK, Halliday WR, Rugg D and Jany CS 2002.Potency, spectrum and residual activity of four new insecticidesunder glasshouse conditions. Florida Entomologist 85(4): 552- 5622 Chawla R.P. and Joia B.S. 1992. Studies on the development ofresistance in diamondback moth (Plutella xylostella L.) toquinalphos in Punjab. J. Insect Sci. 5: 106-108.3 Finney, D.J. 1971. Probit Analysis: A Statistical Treatment of theSigmoid Response Curve. Cambridge University Press, Cambridge,333 p4 Georghiou G.P. 1990. Overview of insecticide resistance. P 19-41. InM.B. Green, H.M. LeBaron and W.K.K. MoBerg (eds). ManagingResistance to Agrochemicals. From Fundamental Research toPractical Strategies, ACS, Washington, DC, USA,5 Hama H. 1989 Characteristic of development of pyrethroid resistance indiamond back moth. Bull. Chugoko Ntl Agril. Expt Station 1989, 4:119-139.6 Joia B.S., Udeaan A.S. and Chawla R.P. 1996. Toxicity of cartaphydrochloride and other insecticides to multi-resistant strains of thediamondback moth, in Plutella xylostella Internat Pest Contr 38:258-597 Krishnamoorthy A. 2002. Biological control of Plutella xylostella (L.),an Indian scenario with referecne to past and future strategies.http://dbm.2002.cirad.fr/papers/krishnamorthy.doc8 Lancaster R and Burt J. 2001. Cauliflower production in WesternAustralia, Bulletin 4521.http://www.agric.wa.gov.au/agency/pubns/bulletin/bull4521/bull4521.pdf9 Mau RFL and Gusukuma-Minuto L 1999. Development of sustainablemanagement program for diamondback moth on cruciferous cropsin Hawaii. Workshop of a integrated pest management of colecrops. I. Insecticide evaluation. Proceedings of the InternationalWorkshop on Integrated Management of Cole Crops. University deCelaya, Guanjuato, Mexico, May 20-21, 1999, pp. 13-23.10 Mau RFL and Gusukuma-Minuto L 2002. Diamnondback mothresistance to spinosad (Success and Tracer, Dow AgroSciences) inHawaii: Confirmation, review of causal factors, and establishmentof a mitigation plan. http://www.inifap-gto.net/Ronaldo.PDF1111 Mehrotra K.N. and Phokela A. 2000. Insecticide resistance in insectpests: Current status and future strategies. In (eds) G.S. Dhaliwaland Balwinder Singh. Pesticides and Environment: 39-85.12 Solang UK and Sribhuddachart J. 2002. Resistance management inPlutella xylostella on ccrucifers in southern Asia.http://www.msstate.edu/Entomology/v7n2/art14.html13 Tabashnik B.E., Cushing N.L. and Finson N. 1987. Leaf residue vstopical bioassay for assessing resistance in the diamondback moth(Lepidopetra: Plutellidae). FAO Pl. Prot. Bull. 35: 11-14.14 Talekar N.S. and Shelton A.M. 1993. Biology, ecology andmanagement of diamondback moth. Ann. Rev. Entomol. 38: 275-301.15 Umeda K., MacNeil D. and Roberts D. 2000. New insecticides fordiamondback moth control in cabbage. The University of ArizonaCollege of Agriculture 2000 vegetable report.http://ag.arizona.edu/pubs/crops/az1177/16 Zhao JZ., Zhu GR and Ju Z. 1995. Efficacy of fipronil anddiafenthiuron on the diamondback moth. Chinese vegetables. No. 2:25-27.http://www.nysaes.cornell.edu/ent/faculty/shelton/Zhao/publication.html (2002)BS Joia, KS Suri, & AS UdeaanDepartment of EntomologyPunjab Agricultural UniversityLudhiana, 141004IndiaArthropod ResistanceResurgence of Spider Mite Tetranychus ludeni Zacher (Acarina: Tetranychidae) Against Acaricides andBotanical Pesticides on CowpeaABSTRACT During the summer months, spider mite(Tetranychus ludeni Zacher) is a detrimental pest ofvegetable crops, especially on cowpea in the Varanasiregion of India. Field experiments were conducted tofind out the resurgence of this mite pest against someacaricides (viz., dicofol (18.5% EC), dicofol (5%WP),abamectin (1.9%EC), phosalone (35%EC), ethion(50%EC), and sulphur (80%WP)), and botanicalpesticides (viz., pongamia oil (2%), N.S.K.E. (5%),neem oil (2%), azadirachtin (0.03%), mahua oil (2%),and PSKE (5%)) at their recommended doses oncowpea crops. The chemical solutions were preparedjust before each spray, and were sprayed every twoweeks. The mortality of spider mite was observed atdifferent intervals including pre-spray and 1, 3, 7, and14 days after spray. The last observation of theprevious spray was counted as the pre-sprayobservation of the following spray, with the rest of theobservations taken similarly. The results indicated thatno resurgence was observed with dicofol (18.5%EC),dicofol (5%WP), abamectin (1.9%EC), and phosalone(35%EC), whereas some resurgence was observed inethion (50%EC) and sulphur (35%EC) despiteencouraging performances. Resurgence was shown in32

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2mahua oil (Madhuca indica), PSKE, pungum oil(Pomgamia pinata), neem oil, azadirachtin, and NSKE.The maximum resurgence was observed with mahuaoil, and the maximum negative resurgence wasobserved with dicofol (18.5 E.C.) in both sprays.Botanical pesticides had some resurgence but could beused as safe pesticides on vegetables, while also beingecologically friendly.KEY WORDS: Resurgence, spider mite, acaricides,botanical pesticides.INTRODUCTION In recent years spider mite, Tetranychusludeni Zacher, has been observed as a serious pest tovegetable crops, especially on cowpea, a commonvegetable of summer months in India. This hasattracted the attention of growers and acarologists inIndia's Varanasi region. Heavy populations of mitesand their profuse webs cover the plants. The regularmonoculture of cowpea without crop rotation hasaggravated the problem. It has been reported thatregular use of dimethoate (Singh et.al 1989) has alsoresulted in outbreaks of this mite. It had been earlierreported that phosphamidon, fluvalinate, fenvalerate,and dimethoate showed resurgence of this mite (Kumarand Singh, 1999). For the economic harvest of thecowpea crop, the current effective treatment is to useacaricides that can manage this mite pest.MATERALS and METHODS Field experiments wereconducted to find out the resurgence of this mite pestagainst some acaricides and botanical pesticides at theirrecommended doses on cowpea crops. The trials werereplicated four times at vegetable grower fields duringsummer months in RBD. The plot size was 3x5m androw-to-row spacing was maintained at 50 cm apart.Twelve formulations of acaricides and botanicalpesticides were taken in this trial (Tables 1 and 2). Thecontrol was treated with water + sandovit spray. Theamount of proprietary ingredient required wascalculated by using the following formula:The observations were taken from five randomlyselected tagged and numbered plants from each plot.Five leaves were plucked from the upper, middle, andlower portions of each plant and a total number oftwenty-five leaves were collected from each plot forobservation. The mite population was counted on thebasis of 2cm squared leaf areas at four spots per leaf.The mortality of spider mite was observed in differentintervals at pre-spray and at 1, 3, 7, and 14 days after.The fourteenth day observation of the first spray wastreated as the pre-spray observation of the 2nd spray,with the rest of the observations taken similarly. Thepercent mortality was calculated by using followingformula:The corrected percent mortality was calculated throughAbbot's formula (1925), which is as follows:Where:P = percent corrected mortality,P1 = percent observed mortalityC = percent mortality in control.The percent resurgence of mite population wascalculated by Henderson and Tilton's formula asfollows:Where:Ts = Number of live mite in posttreatment countTF = Number of live mite in pretreatment countCs = Number of mite in untreatedcheck (Post-treatment)CF = Number of mite in untreatedcheck (Pre-treatment)RESULTS and DISCUSSION The results indicate that somebotanical pesticides showed maximum positiveresurgence viz. mahua oil (+ 38.68 %), neem oil (+12.26 %), PSKE (+ 10.36 %), NSKE (+ 5.22 %), andpongamia oil (+ 4.68 %) (Table 1). Some acaricidesviz. sulphure (+ 6.22 %), ethion (+ 3.72 %), andbotanical pesticides i.e. azadirachtin (+ 4.66 %) hadencouraging performances but showed someresurgence, whereas resurgence was not noticed withdicofol 18.5% EC (- 26.28 %), dicofol 5 % WP (-14.22 %), abamectin (- 12.22 %), and phosalone (-10.68 %) in the first spray (Table 1). In the secondspray, the trend of maximum resurgence of somebotanical pesticides was changed, including mahua oil(+ 88.64 %), pongamia oil (+ 32.66 %), P.S.K.E. (+25.00 %), neem oil (+ 18.43 %), azadirachtin (+ 14.09%), and NSKE (+ 8.32 %) (Kumar & Singh (1998) andRai et al. (1993) supported these findings). There wasno observed change in the trend of the encouragingperformance of acaricides viz., sulphur (+ 8.32 %), andethion (+ 7.99 %) (Table 2). The same trend wasfollowed in the second spray. No resurgence wasshown with some acaricides like dicofol 18.5%EC (-74.30 %), dicofol 5% WP (- 21.65 %), abamectin (-18.02 %), and phosalone (- 12.75 %) (Table 2).Zhang and Sanderson (1990) earlier reported therelative toxicity of abamectin to Phytoseiuluspersimilis Anthias-Henriot and the spider mite33

Tetranychus urticae Koch. At 24 hrs aftertreatment, two spotted spider mites survived but mostwere immobilized at concentrations of 1, 4, and 8 ppm;at 16 ppm, two spotted mite mortality was >70% aftertreatment. Survival and mobility of two spotted spidermites were significantly affected at high concentrationsof 4 and 8 ppm (almost all survivors wereimmobilized). Singh and Singh (1992) have reportedthe effectiveness of dicofol against this mite. Dicofoland phosalone were statistically on par with untreatedcontrols but superior to the rest, indicating that they didnot induce any mite resurgence but rather becameineffective against the mite.Kumar and Singh (1999) had earlier reported thatthe menace of spider mite, Tetranychus urticae Koch,has been identified as major problem of okra duringsummer months. In this experiment, neem-basedformulations and conventional acaricides were used forthe study of resurgence. The results indicated thatphosphamidon, fenvalerate, and dimethoate showedresurgence of spider mites. The neem-basedformulation (azadirachtin) showed encouragingperformance but showed resurgence.Kumar et al. (2001) reported that some pesticidesshow resurgence of two spotted mite viz.,phosphamidon + 128.51% and dimethoate + 118.18%,while azadirachtin shows encouraging performances +14.89 % resurgence. Resurgence was not noticed byacaricides viz., dicofol - 67.78 % and phosalone - 16.06%.Kumar et al. (2002) reported that no resurgencewas observed with dicofol (EC), dicofol (WP),abamectin, and phosalone, whereas encouragingperformance but some resurgence was noticed withethion (50%EC) and sulphur (80%WP). However,resurgence in mahua oil, PSKE, pungum oil, neem oil,azadirachtin, and NSKE did not show any effectivenessagainst this pest.REFERENCESKumar, Sunil and, R.N. Singh. 1998. Bio-efficacy of neem pesticide onTetranychus urticae Koch as compared with some conventionalacaricide. ANACON-98, Mumbai, Abstract. 6.Kumar, Sunil and R.N. Singh. 1999. Studies on resurgence of spider mite(Tetranychus urticae) on okra, National Symposium on Biologicalcontrol of insects in Agriculture, Forestry, Medicine and VeterinarySciences. Abstract and Souvenir. 9.Kumar, Sunil; Surendra Prasad, and R.N. Singh. 2001. IntegratedManagement of Two spotted mite Tetranychus urticae Koch onokra crop. Symposium on Biological control Based Managementfor Quality Crop Protection in the Current Millennium. Proceedingof symposium, July 18-19, PAU, Ludhiana, India, 193-194.Kumar, S.; S. Prasad, and, R.N. Singh. 2002. Resurgence of Two SpottedMite, Tetranychus urticae Koch. (Acarina: Tetranycidae) due toAcaricides and Botanicals on Okra. Ann.Pl. Protec. Sci. 10 (2): 239-242.Rai, A. B.; G. R. Bhanderi and C. B. Patel. 1993. Effectiveness of neembasedproducts (Azadirachtin) against Tetranychus macfarlanei(Tetranychidae) on okra (Abelmoschus esculentus L.). Moench andtheir safety to predatory mites. Neem Newsl. 10 (1-2): 26.Singh, R. N.; I. N. Mukherjee; R. K. Singh and J. Singh. 1989. Evaluationof some pesticides against Red spider mite, Tetranychus ludeniZacher on cowpea. The first Asia Pacific Conference ofEntomology, 566 -569.Singh, R.N. and J. Singh. 1992. Evaluation of some pesticides againstcarmine mite, Tetranychus cinnabarinus (Boisd.) on Lady's finger.Pestology. 16 (2): 20-23.Zhi-Qiang Zhang and John P. Sanderson. 1990. Relative Toxicity ofAbamectin to the Predatory Mite Phytoseiulus persimilis (Acari:Phytoseiidae) and Two spotted Spider Mite (Acari: Tetranychidae),Journal of Economic Entomology 83(5), 1783-1790.S. Kumar, S. Prasad & R. N. SinghUPCAR- MITE PROJECTDepartment of Entomology & Agricultural ZoologyInstitute of Agricultural SciencesBanaras Hindu UniversityVaranasi - 221 005IndiaInsecticide Usage Patterns in South Indian Cotton Ecosystems to Control Cotton Bollworm, HelicoverpaarmigeraCotton, as an important commercial crop of India,has boosted the economic conditions of farmersespecially when introduced under irrigation. Thetraditional cotton ecosystem of South India is spreadover Maharastra, Andhra Pradesh, Karnataka, andTamil Nadu where the well-known block cotton soilshave prevailed, both under assured irrigation andrainfed situations. A complex of insect pests in Indiahas damaged cotton and pulse crops. The cottonbollworm, Helicoverpa armigera, has been a majorpest with severe economic consequences and hasinflicted huge crop losses ranging from 47-90%. As aresult, cotton crops grown over only 5% of the totalcultivated area have consumed more than 55% of thetotal amount of pesticides used in India (1). The cottonbollworm has attained the status of a national pestowing to its devastative nature on cotton and othercrops in India and elsewhere. Frequent outbreaks ofHelicoverpa armigera in India on cotton crops havelead to severe social disturbances, with several reportsof suicide by farmers (2). To combat the unprecedentedH. armigera pressure, farmers in the region haveapplied synthetic pyrethroids or organophosphateinsecticides - sometimes as mixtures at 2-3 dayintervals - during critical periods, resulting in over 30sprays (against the recommended 8-10 sprays). Thishas led to the development of high levels of insecticideresistance in the cotton ecosystem (1). During 1992-1997, crop failure in many states of the South Indiancotton ecosystem, particularly Andhra Pradesh andKarnataka, was followed by the suicide of several

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2farmers, which has been traced to insecticide resistancein H. armigera (3).The consumption pattern of different insecticidesbelonging to different groups varies across thegeographic locations primarily based on the dealerrecommendations, intensity of pests and diseases,influence of peer groups, efficacy of particularinsecticides, knowledge level of the farmer, availabilityof a particular insecticide, and socioeconomicconditions of the farmer (4). Though a number ofstudies were conducted on knowledge and pesticideuse, the changing scenario warrants more studies. Inview of this, to determine the relative predominance ofindividual insecticide molecules and their relativeusage over the South Indian cotton ecosystem, a surveywas undertaken during the 2000-2001 cropping season.We selected four South Indian states viz.,Maharastra, Andhra Pradesh, Karnataka, and TamilNadu, which comprise more than 95 percent of thecotton cultivation in South India. Looking into thedistribution of cotton cultivation, three samplinglocations in each state were selected to collect the data.The locations selected included: Nagpur, ParbhaniNanded (Maharastra), Guntur, Madhira, Kovilpatti(Andhra Pradesh), Raichur, Dharwad, Mysore(Karnataka), Coimbatore, Madurai, and Kovilpatti(Tamil Nadu). During the cropping season, eachlocation was visited and interacted with by at least 25farmers with a schedule on various aspects of insectpest control including insecticide usage pattern.Aspects concerning insecticides being used, dosage perapplication, number of insecticide applications percrop, number of times a particular insecticide was used,and the relative efficacy in farmers' perception etc.,were collected. Wherever possible, fields were visitedand actual prevalence of cotton bollworm was studied.Later, information on number of insecticides, relativeusage in terms of number of sprays, concentration,farmer perception, and attitude, etc., were computed foreach location and overall cotton ecosystem of SouthIndia.Overall, in the South Indian cotton ecosystem, asmany as 15 different insecticides: monocrotophos,quinolphos, chlorpyriphos, cypermethrin, decamethrin,acephate, endosulfan, fenvelarate, polytrin, sumicidine,carbaryl, permethrin, avaunt, Bacillus thurengenesis(Bt), and spinosad were used with the specificobjective of controlling cotton bollworm. Amongchemical insecticides, monocrotophos was the mostextensively used insecticide with a share of 26.8percent of all the insecticides, followed bychlorpyriphos (19.9%), quinolphos (18.8%),cypermethrin (14.93%), and endosulfan (12.53%)(Figure 1). Bt was the only biological agentencountered in the whole ecosystem and accounted for0.76% overall. However, it was used only in theRaichur region of Karnataka state where it forms9.09% of all the insecticides used in the region.Similarly, spinosad (a recent chemical in South Indiawhich is not yet recommended and commerciallyavailable in market), permethrin, and polytrincomprised 0.36%, 0.46%, and 0.76% respectivelyoverall in the ecosystem but were used only inCoimbatore (4.34%), Nalagonda (5.55%), and Nanded(9.09%) respectively (Table 1). The number ofinsecticides being used to control bollworm variedacross locations in South India. A maximum of 8insecticides including 1biological agent were recordedin the Raichur region followed by 7 in Nalagonda,Coimbatore, and Kovilpatti. Farmers in the Mysoreregion used 4 different insecticides, which was the leastnumber in the overall ecosystem. Raichur is knownhistorically for being the cotton city of India and for itshigh intensity use of insecticides in Asia. Apart fromusing the maximum number of insecticides, Raichurrecorded up to 25 sprays to control the bollworm in thepresent study. Most of the cotton regions of northernstates of South India used between 18-20 sprays, whileDharwad and Mysore used fewer (8-12 and 10-12)numbers of applications of insecticides in the region(Table 1). In all of the locations the usage ofinsecticides was erratic and indiscriminate. Overall, 60-75% of the farmers applied the insecticides as mixtureof 3 to 6 in an interval of 2-3 days during the criticalperiod. Armes et al (5) reported similar insecticideusage patterns in Karnataka and Andhra Pradesh forthe control of H. armigera. Generally, only the firsttwo sprays were not mixtures of insecticides, andmonocrotophos was always used throughout SouthIndia without exception. However, only 21.23% ofcotton farmers of the Raichur region used theinsecticides as a mixture. It was interesting to note that78.77% of the farmers in this area used a definiteschedule of insecticides for the control of cottonbollworm. Most of the farmers in this region rotatedthe insecticides. Further, this was only region where36

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2credit. The dealers profited from usingthe illiteracy of the farmers, and the lackof a good extension services in many ofthe remote rural areas also aggravatedthe problem. Prolonged use of the sameinsecticides will definitely elevate theproblem of insecticide resistance, as hadhappened in Andhra Pradesh andKarnataka during late 1980s and early1990s (1).Identification of insecticide usagepatterns allows for the rotation ofinsecticides and the rational use ofpesticides for the better management ofthe pest and for insecticide resistance (4,7). This study brings to light that farmerknowledge about insecticide usage isvery poor and as such, insecticides arebeing used indiscriminately by thefarmers were aware of and used Bt for the control ofbollworm on cotton.Monocrotophos was the single most commoninsecticide used in all of the locations of South Indiafor the control of cotton bollworm. In all of the regionsexcept Kovilpatti and Nalagonda, monocrotophos andquinolphos were the primary choices, by more than 30percent, of insecticides for use in controlling bollworm.Both the relative prevalence and the use of insecticidesvaried across the geographical locations of South India(Table 2).It was very clear from the survey that the majorityof the farmers were greatly influenced by the dealers.Beside the fact that the pesticide dealers had such animpact on the pesticide use pattern among farmers, thefarmers tended to be more loyal to those dealers whoalso provided technical advice in all aspects of plantprotection. These results were in line with those ofRakila and Padmanaban (6). The main reason for thisdependence appeared to be that most farmers wereeconomically poor and depended on the dealers forfarmers of South Indian cotton ecosystems. Theexceptions are the regions of Raichur and a few regionsof Andhra Pradesh, which suffered severe outbreaks inthe past. Perhaps the extensive extension efforts withrespect to insecticide usage in these regions areresponsible for the better knowledge of the farmers (7).In order to rationalize the pesticide use on the farms, itis imperative to stress the importance of economicthreshold levels in the application of pesticides and tofollow the integrated pest management practices tobring down the expenditure and to increase theeffectiveness of plant protection measures in cotton.Further, the outcome of the survey clearly indicates theneed for genetic investigations of the geographicpopulations of bollworm and the formulation ofpopulation specific integrated pest management (IPM)modules. Based on the genetic similarity and theinsecticide composition patter by different geographicpopulations, we need to force the rotation of modulesfor the better management of cotton bollworm andinsecticide resistance. Concomitantly, there is a greater37

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2need for educating the farmers about the pests,insecticides, and their uses to avoid indiscriminateusage and to prevent a chain of problems that effectnature and human health.REFERENCES:1. Kranthi KR , Jadhav DR, Wanjari RR, Shakir Ali S Russel D.Carbomate and organophosphate resistance in cotton pests in India,1995 to 1999. Bull. Ent. Res., 2001; 91:37-46.2. Patil B.V, Lingappa S, Srinivasa A.G, Bheemanna M. Integrated pestManagement Strategies for Cotton. Paper presented at XXinternational congress of Entomologyheld at Firenze, Italy from 25-31, August 1996.3. Kranthi KR, Armes NJ, Nagarjun GV, Rao RS, Sundaramurthy VT.Seasonal dynamics of metabolic mechanisms mediating pyrethroidresisitance in Helicoverpa armigera in central India. Pestic. Sci.,1997;50:91-98.4. Lingappa S, Panchabhavi KS, Hugar PS. Managament of cottnbollworms with special reference to Heliothis in Karnataka.Petology, 1993;17(9):20-25.ACKNOWLEDGEMENT: The financial assistance in theform of a project by Dept. of Biotechnology (DBT),Govt. of India is gratefully acknowledged by Dr. B.Fakrudin. We are also grateful to all the scientists at allthe sampling locations in South India who helped invarious ways during our visit.5. Armes NJ, Jadhav DR, Bond GS, King ABS. Insecticide resisitance inHelicoverpa armigera in South India. Petic. Sci. 1992:34:355-364.6. Rakila A, Padmanabhan NR.. Knowledge and factors influencingpesticide use and frequency of plant protection measures.Pestology,1995;19: 9-127. Jadhav DR, Armes NJ. Comparitive status of insecticde resisitance inHelicoverpa and Heliothis species (Lepidoptera: Noctuidae) ofSouth India. Bull. Ent. Res. 1996; 86:525-531.B. Fakrudin, B. V. Patil, P. R. Badari Prasad, & S. H.PrakashDepartment of BiotechnologyUniversity of Agricultural SciencesDharwadIndiaRecent Advances in Host Plant Resistance to Whiteflies in CassavaINTRODUCTION Whiteflies are considered one of theworld’s major agricultural pest groups, attacking awide range of plant hosts and causing considerablecrop loss. There are nearly 1200 whitefly species witha host range that includes legumes, vegetables, fruittrees, ornamentals, and root crops. As direct feedingpests and virus vectors, whiteflies cause major damagein agroecosystems based on cassava (Euphorbiaceae;Manihot esculenta Crantz) in the Americas, Africa, andto a lesser extent Asia. The most damaging species oncassava in northern South America is Aleurotrachelussocialis. Typical damage symptoms include curling ofapical leaves, yellowing and necrosis of basal leaves,and plant retardation (Fig. 1). Adult whiteflies are mostfrequently observed on the underside of apical leaveswhere they feed on plant fluids and oviposit. The"honeydew" excreted is a substrate for a sooty-moldfungus that interferes with photosynthesis (Fig. 1C).The combination of direct feeding and impairedphotosynthetic rate reduces root yield by 4% to 79%depending on the duration of attack (Bellotti, 2002).More than 5,000 cassava genotypes have been38

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2evaluated at CIAT and CORPOICA for whiteflyresistance. At present, the major source of hostresistance in cassava is the genotype MEcu-72 (Bellottiand Arias, 2001) (Fig. 1D). When feeding on MEcu-72A. socialis had less oviposition, longer developmentperiods, reduced size, and higher mortality than whenfeeding on the susceptible genotype (Fig. 2). Due to theimportance of whiteflies as a pest and virus vector, it isimportant to understand the nature of genes that conferresistance in the resistant genotype, MEcu-72. To studythe genetics of this resistance, a cross was madebetween MEcu-72 (resistance genotype) x MCol-2246(a very susceptible genotype), to evaluate F1segregation, using molecular markers. This willaccelerate the selection of whitefly resistant germplasmand isolate resistant genes.MATERIALS and METHODSPlant Material: For the present work we have used thecross MEcu-72 (as the resistant parent) x MCol-2246(as the susceptible parent). A total F1 offspring of 286genotypes (family CM8996) was produced from thiscross. These materials were sowed and evaluated in thefield during May 2001, March and August 2002 at twodifferent locations: Espinal-Tolima, Colombia(CORPOICA-NATAIMA) at 350 m.a.s.l. andSantander de Quilichao, Cauca, Colombia, at 990m.a.s.l. With this evaluation we will identify genesegregation in the offspring and we will be able toselect the resistant and susceptible materials. Theevaluation was performed in the field using populationand damage scales ranging between 1-6, where 1 is anabsence of damage and population and 6 is highpopulation and damage(curling, chlorosis, sooty moldfungus, etc.) (Table 1). Threeevaluations were performed in2002 using the highest damageand population data forinformation processing.genome equivalents. The whitefly resistance will betarget for map-based cloning using the BAC librariesas tools. We are using silver staining to visualize theallelic segregation of the markers.RESULTSField Evaluation: The whitefly resistant variety CG489-31 (Fig. 2), a progeny from the MEcu 72 x MBra12 cross, has been released to cassava farmers byCORPOICA, Colombia under the name Nataima-31(Fig. 3).Initial field evaluations showed that thesematerials (family CM 8996) had low levels of the pest,because test plants (materials very susceptible to A.socialis) did not present high levels of damage andpopulations (scale of 4 to 6 Table 1). The harvestevaluation showed that the root yield was between 4.5and 86.5 ton/ha, and many materials presenteddesirable characteristics (high percentage of dry matter,palatability, etc.). Currently, the family is under asecond sowing cycle at the same locality from Tolima,and high pressure exerted by the pest has been detectedsince test materials have high degrees of damage (from4 to 5).Preliminary evaluations have demonstrated thatsome materials from the family present low levels ofdamage and population (up to 2) (Fig. 4 and 5).Molecular Analysis: Both parents MEcu-72 and MCol-2246 were evaluated with 343 cassava SSR markers(Mba et al, 2001) including 156 cDNA SSRsdeveloped (Mba et al, by submitted). Approximately155 of the SSRs were polymorphic in the parentals andMolecular Analysis: We areusing Simple Sequences Repeat(SSR) to find markersassociated with resistance formapping the resistant gene(s).As part of a collaborativeproject with ClemsonUniversity funded by USAID aBAC library for cassava usingthe clone MEcu 72 wasconstructed. The librarycontains 73,728 clones with anaverage insert size of 93 kb.Based on a genome size of 760Mb, library coverage isapproximately 10 haploid39

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2were evaluated in the F1 (Fig. 6).Association between Molecular Markers andResistance: The molecular data are being analyzedusing QTL packages (QTL cartographer Qgene) todetermine linkages between the markers and thephenotypic characterization. As preliminary analysis X2 at the 5% level was done using SAS. Putativeassociations were found between 43 SSRs markers andthe field phenotypic characterization (score 1.0 to 2.0of the levels of damage and populations).CONCLUSIONS and ONGOING WORKThe field evaluations in the family CM 8996 andtheir parental show the resistance of the genotypeMEcu-72 and the high susceptibility of theparental MCol-2246.Using SSR markers, putative association with theresistant lines was found. A linkage map is beingconstructed using the SSR data and the fieldphenotypic characterization.Based on going QTL analysis, the marker linked tothe resistant gene(s) will be used as part of largescale screening of breeding lines and to acceleratethe breeding cycle for whiteflies resistance. Finemapping of the genes involved will be carried as afirst step toward the cloning of the resistant genesand the study of their expression.ACKNOWLEDGEMENTS We acknowledge the valuablecontribution of Nelson Morante in the establishment ofplants in the field.The New Zealand ministry of foreign Affairs andTrade provided financial support for parts of theresearch carried out in this work.40

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2REFERENCESArias B. 1995. Estudio sobre el comportamiento de la “Mosca Blanca”Aleurotrachellus socialis Bondar (homoptera: Aleyrodidae) en diferentesgenotipos de Yuca, Manihot esculenta Crantz. Thesis.Bellotti, A.C. and B. Arias, 2001. Host plant resistance to whiteflieswith emphasis on cassava as a case study. Crop Protection 20:813- 823.Bellotti, A.C. 2002. Arthropod Pests: In Cassava: Biology, Productionand Utilization (Eds. R.J Hillocks, J.M. Thresh and A.C. Bellotti. CABInternational 2002. 332pp.Brown, J. K., D. R. Frolich, and R. C. Rosell. 1995. The sweet potato orsilver leaf whiteflies: biotypes of Bemisia tabaci or a species complex.Annu. Rev. Entomol. 40: 511-534.CIAT, 1995. Cassava program annual report. 54p.Fregene, MA, Angel, F, Gomez, R, Rodriguez, F, Roca, W, Tohme, J.and M. Bonierbale. 1997. A molecular genetic map of cassava (Manihotesculenta Crantz). Thor. Appl. Genet. 95: 431-441.41

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Mba, R.E.C., Stephenson, P., Edwards, K., Melzer, S., Mkumbira, J.,Gullberg, U., Apel, K., Gale, M., Tohme, J. and Fregene, M. 2001.Simple Sequence Repeat (SSR) Markers Survey of the Cassava(Manihot esculenta Crantz) Genome: Towards an SSR-BasedMolecular Genetic Map of Cassava. Theoretical and AppliedGenetics Journal. 102:21-31.A. Bellotti, B. Arias, A. Bohorquez, J. Vargas,G. Trujillo, C. MBA, M. C. Duque, & J. TohmeCentro Internacional de Agricultura Tropical (CIAT)AA6713, CaliColombiaandH. L. VargasCORPOICAEspinal, TolimaColombiaEvidence for Multiple Mechanisms of Resistance to Cry1Ac and Cry2A Toxins from Bacillus thuringiensis inHeliothis virescensBacillus thuringiensis (Bt) is a common sporeformingbacterium that produces insecticidal proteinscalled Cry toxins (from Crystal). Thecommercialization of transgenic plants producing Crytoxins has greatly affected insect control methods dueto their environmental safety and increased crop yield.In 1996, transgenic cotton plants producing Cry1Actoxin were commercialized to control Heliothisvirescens (tobacco budworm). This insect is one of themost important pests of cotton, among other crops. Aswith any insect control method, development ofresistance to Bt toxins is one of the main concerns onthe wide use of transgenic Bt plants. Although no H.virescens resistance episodes to Bt cotton have beenreported in the field so far, laboratory resistanceselection of H. virescens has demonstrated that thegenetic potential for resistance development exists(Gould et al., 1992, 1995). The study of resistance inthese laboratory-selected insect strains has helped toidentify potential resistance mechanisms and strategiesaimed to manage and delay the onset of resistance.Disruption of any step in the mode of action of Bttoxins can result in resistance to these toxins. Thegeneral mode of action of Bt toxins includes ingestionby the susceptible insect, solubilization and activationto toxic forms by insect midgut enzymes, binding andinsertion into the membrane of the midgut epithelium,and midgut cell lysis by osmotic shock (Knowles,1994). Although several mechanisms of resistance toBt toxins in laboratory-selected insects have beenproposed, alteration of toxin binding to midgutreceptors is the best studied (Ferré and Van Rie, 2002).Since an insect is less likely to develop resistanceto two toxins with distinct modes of action, one of theproposed methods to delay the onset of resistance to Btplants in the field is the generation of transgenic linesexpressing different Bt toxins in combination (Gould,1998). To assure the efficacy of this approach thetoxins selected for expression should not sharecommon binding sites and must have distinct modes ofaction.In brush border epithelium membrane vesicles(BBMV) from H. virescens, Cry1Aa, Cry1Ab, Cry1Ac,Cry1Fa, and Cry1Ja toxins share a common bindingsite (receptor A). Cry1Ab and Cry1Ac have anadditional binding site (receptor B) and Cry1Ac is theonly toxin that can recognize a third binding site(receptor C) (Van Rie et al., 1989; Jurat-Fuentes andAdang, 2001). According to this model of bindingsites, alteration of receptor A would potentially lead toreduced binding and possibly resistance to all Cry1A,Cry1Fa and Cry1Ja toxins. This mechanism wasproposed to occur in the Cry1Ac-selected YHD2 strainof H. virescens (Lee et al., 1995).One of the most important toxin candidates to beused in combination with Cry1Ac in Bt cotton tocontrol H. virescens is Cry2A. This toxin does notshare binding sites with Cry1A toxins (Jurat-Fuentesand Adang, 2001) and has a distinct mode of action(English et al., 1994; Morse et al., 2001). TransgenicBt cotton plants expressing both Cry1Ac and Cry2Ahave been shown to enhance control of H. virescens(Stewart et al., 2001). Interestingly, the Cry1Aclaboratory selected CP73-3 and KCB H. virescensstrains developed cross-resistance to Cry2A, amongother toxins (Gould et al., 1992; Forcada et al., 1999).These strains were backcrossed to susceptible insectsand the offspring were selected with Cry2A to increaseresistance to this toxin. This selection regime led to thegeneration of the CXC (derived from CP73-3) andKCBhyb (derived from KCB) strains, which showedincreased Cry2A and Cry1Ac resistance levels whencompared to their parental strains (Kota et al., 1999).Both strains were also cross-resistant to Cry1Aa,Cry1Ab, and Cry1Fa toxins (F. Gould, unpublishedobservation).To study the mechanism of resistance in the CXCand KCBhyb strains, we performed toxin-bindingassays with radio labeled Cry1A toxins. BBMV fromYDK (susceptible control strain), CXC and KCBHybinsects were isolated and incubated with increasingconcentrations of labeled Cry1A toxins to generate abinding saturation curve for each Cry1A toxin.42

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Saturation curves were analyzed and the bindingaffinities of each toxin for the CXC, KCBhyb, andcontrol susceptible BBMV were calculated. Nochanges in either toxin affinity or concentration ofreceptors were detected in BBMV from the CXC strainwhen compared to susceptible vesicles. On the otherhand, binding of Cry1Aa was greatly reduced invesicles from KCBhyb, while Cry1Ab and Cry1Acbinding was as in BBMV from susceptible insects.These results are evidence that resistance in theCXC strain is not due to changes in toxin binding tomidgut receptors. Resistance in this strain should be theresult of a change in a common step of the Cry1Ac andCry2A toxin mode of action. Since these toxins seemto recognize different receptors in H. virescens, onepossibility is alteration of steps prior to receptorbinding in this strain. Such a change in thesolubilization or processing of the Cry toxins inmidguts of CXC insects would lead to resistance toboth Cry1Ac and Cry2A. The existence of such amechanism would be consistent with the decreasedlevels of susceptibility to other Bt toxins, as is the casefor Cry1Aa, Cry1Ab, and Cry1Fa.Since Cry1Aa and Cry1Fa share a commonbinding site, we used biotinylated Cry1Fa (sinceiodination inactivates this toxin) to study binding ofthis toxin to BBMV from KCBhyb. No differences inCry1Fa toxin binding were observed between YDKand KCBhyb, suggesting that binding of this toxin isnot altered in KCBHyb larvae. Or at least, Cry1Fabinding is not altered to a degree detectable by thebinding assay. Since Cry1Aa shares its only BBMVbinding site with Cry1Ab, Cry1Ac, and Cry1Fa, thechange that is preventing Cry1Aa binding in KCBhybBBMV is probably also responsible for resistance to allthese toxins. This hypothesis was also proposed for theCry1Ac resistant YHD2 strain of H. virescens (Lee etal., 1995) after obtaining the same qualitative toxinbinding results we observed in KCBHyb BBMV.Additionally, since Cry1Aa and Cry2A do not sharebinding sites in H. virescens BBMV, cross-resistanceto Cry2A cannot be explained by alteration of Cry1Aabinding. In this case, a second mechanism of resistancethat would affect both Cry1Ac and Cry2A mode ofaction needs to be present. As outlined for the CXCstrain such a mechanism is may be related to alterationof toxin solubilization and/or processing conditions inthe midguts of CXC and KCBhyb midguts.In conclusion, our results indicate the presence ofat least two resistance mechanisms in larvae from theKCBHyb strain. One of the mechanisms would berelated to Cry1A receptor alteration, and possibly thesecond mechanism related to toxin solubilizationand/or processing in the larval midgut. Similarconclusions have been presented for resistant Plodiainterpunctella (Indianmeal moth) (Herrero et al., 2001).Alteration of toxin solubilization and/or processingseems to be the main mechanism of resistance in larvaefrom the CXC strain. Interestingly, high levels ofCry2A expression in chloroplasts of tobacco plantsovercome resistance in CXC larvae (Kota et al., 1999),indicating a possible solution to this resistancemechanism. Nevertheless, our conclusions raisequestions as to how H. virescens in the field willrespond to transgenic cotton producing Cry1Ac andCry2A proteins. Our results are also evidence of thearray of resistance mechanisms to Bt toxins that H.virescens can develop after selection with a single Crytoxin. This information is extremely important whendesigning and implementing strategies aimed atdelaying resistance and cross-resistance to Bttransgenic crops.Experiments in our laboratory are presently aimedat elucidating the molecular mechanism by whichdecreased toxin binding is achieved in the KCBhybresistant insects, as well as the molecular nature of theresistance mechanism in CXC larvae.REFERENCESEnglish, L., Robbins, H.L., Von Tersch, M.A., Kulesza, C.A., Ave, D.,Coyle, D., Jany, C.S., and Slatin, S. (1994) Mode of action ofCryIIA: a Bacillus thuringiensis delta-endotoxin. Insect. Biochem.Molec. Biol., 24, 1025-1035.Ferré, J., and J. Van Rie. 2002. Biochemistry and genetics of insectresistance to Bacillus thuringiensis. Annu. Rev. Entomol. 47, 501-533.Forcada, C., Alcácer, E., Garcerá, M. D., Tato, A., and R. Martínez. 1999.Resistance to Bacillus thuringiensis Cry1Ac toxin in three strains ofHeliothis virescens: proteolytic and SEM study of the larval midgut.Arch. Insect Biochem. Physiol. 42, 51-63.Gould, F., A. Martínez-Ramírez, A. Anderson, J. Ferré, F. J. Silva, andW. J. Moar. 1992. Broad-spectrum resistance to Bacillusthuringiensis toxins in Heliothis virescens. Proc. Natl. Acad. Sci.USA 89: 7986-7990.Gould, F., A. Anderson, A. Reynolds, L. Bumgarner, and W. Moar. 1995.Selection and genetic analysis of a Heliothis virescens(Lepidoptera: Noctuidae) strain with high levels of resistance toBacillus thuringiensis toxins. J. Econ. Entomol. 88: 1545-1559.Gould, F. 1998. Sustainability of transgenic insecticidal cultivars:integrating pest genetics and ecology. Annu. Rev. Entomol. 43,701-726.Herrero, S., Oppert, B., and J. Ferré. 2001. Different mechanisms ofresistance to Bacillus thuringiensis toxins in the Indianmeal moth.Appl. Environ. Microbiol. 67, 1085-1089.Jurat-Fuentes, J.L., Adang, M.J. 2001. Importance of Cry1 d-endotoxindomain II loops for binding specificity in Heliothis virescens. Appl.Environ. Microbiol. 67, 323-329.Knowles, B. (1994) Mechanism of action of Bacillus thuringiensisinsecticidal delta-endotoxins. Adv. Insect Physiol., 24, pp. 275-308.Kota, M., Daniell, H., Varma, S., Garczynski, S. F., Gould, F., and W. J.Moar. 1999. Overexpression of the Bacillus thuringiensis (Bt)Cry2Aa2 protein in chloroplasts confers resistance to plants againstsusceptible and Bt-resistant insects. Proc. Natl. Acad. Sci. USA 96,1840-1845.Lee, M. K., Rajamohan, F., Gould, F., Dean, D.H. 1995. Resistance toBacillus thuringiensis Cry1A d-endotoxins in a laboratory-selectedHeliothis virescens strain is related to receptor alteration. Appl.Environ. Microbiol. 61, 3836-3842.Morse, R. J., Yamamoto, T., and R. M. Stroud. 2001. Structure of Cry2Aasuggests and unexpected receptor binding epitope. Structure 9, 409-417.Stewart S. D., Adamczyk, J. J. Jr, Knighten, K. S., and F. M. Davis. 2001.Impact of Bt cottons expressing one or two insecticidal proteins ofBacillus thuringiensis Berliner on growth and survival of noctuid(Lepidoptera) larvae. J. Econ. Entomol. 94, 752-60.43

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Van Rie, J., Jansens, S., Hofte, H., Degheele, D., Van Mellaert, H., 1989.Specificity of Bacillus thuringiensis d-endotoxins: importance ofspecific receptors on the brush border membrane of the midgut oftarget insects. Eur. J. Biochem. 186, 239-247.Juan L. Jurat-FuentesDepartment of EntomologyUniversity of GeorgiaAthens, GA-30602USAFred L. GouldDepartment of EntomologyNorth Carolina State UniversityRaleigh, NC-27607USAMichael J. AdangDepartments of Entomology andBiochemistry and Molecular BiologyUniversity of GeorgiaAthens, GA-30602USAMonitoring Onion Thrips Resistance to Pyrethroids in New YorkThe onion thrips, Thrips tabaci Lindeman, is a pestof onions and related Allium species, as well as dozensof other plant families, throughout the world. Themajor control strategy for onion thrips on onions is thefrequent use of insecticides and growers may applytreatments weekly, especially in hot, dry years. There isconcern that such intensive treatments may result in thedevelopment of resistance. The results reported in thispaper include studying the susceptibility of onion thripspopulations in New York to l-Cyhalothrin (Warrior T)in 2001 and in 2002 using a newly developed assaysystem and conducting a simulated field test tocompare the results from the laboratory assays to fieldperformance.MATERIALS and METHODSEvaluating Onion Thrips Populations against Warrior:We used the Thrips Insecticide Bioassay System(TIBS) developed by Rueda and Shelton (2003). Thissystem allows thrips to be collected directly from onionplants into a 0.5 ml microcentrifuge tube previouslytreated with an insecticide. Thrips mortality is assessedat 24 h with the help of a dissecting stereoscope andthen data are analyzed to calculate the meanlethal concentration that would kill 50% of thepopulation (LC50). During the 2001 growingseason, thrips populations were collected from 16different sites encompassing the major growingareas of New York. In 2002, populations werecollected from 10 different sites at two differenttime periods (mid-season and late-season). Thereason for the two collections was to determinewhether there are changes in susceptibility overtime. For each of these collections we only testedthe populations against a single dose, the fielddose of 100 ppm. This decision was based onevidence collected in 2001 that fields in which itwas difficult to control thrips with Warrior hadLC50 values > 100 ppm.Determine the Relationship between Lab Assaysand Simulated Field Performance:From our collections of onion thrips in 2002,we selected four populations that varied significantly intheir susceptibility to Warrior, based on our laboratoryassay using TIBS. We introduced these populationsonto onion plants treated with the field rate of Warriorusing a carbon dioxide backpack sprayer. Afterspraying, the insecticide was allowed to dry for 24hours and then 20 thrips larvae were introduced into acage containing a single plant. Thrips were allowed tofeed and then mortality was assessed after 5 days.There were eight cages (replicates) for each of the fourpopulations of thrips used in this study.RESULTS and DISCUSSIONEvaluating Onion Thrips Populations against Warrior:Of the 16 populations examined in 2001, sevenhad LC50 values greater than the field dose of 100 ppmindicating a potential for poor field performance(Figure 1). In the figure the fields are grouped byregion (e.g. Orange County denoted by OR 1-3)) andthere was considerable variation within each regionwith some fields having populations of thrips withLC50 values much higher or lower than the field rateof 100 ppm. This indicates that individual grower44

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2practices probably dictate the developmentof resistance.In 2002, we assessed the susceptibilityof thrips populations based on the %mortality at the field rate of 100 ppm (Figure2). For the first collection (mid-season), therewas considerable variation in the % controlwith half the populations having >50%mortality at 100 ppm. For the late-seasoncollection, there was again considerablevariation in the % control with half thepopulations having > 50% mortality at 100ppm. However, only two of the populationsthat were susceptible (>50% mortality at 100ppm) in the early part of the season weresusceptible in the later part of the season. Wesuspect that changes in insecticide usecaused these changes, but need to examine sprayrecords to determine if this is the case.Determine the Relationship between Lab Assays andSimulated Field Performance:As we had suspected, there was an excellentrelationship (r2= 0.97) between the mortality at 100ppm using the TIBS method and the level of controlthat was seen when onion plants were sprayed with thefield rate of Warrior (100 ppm). This indicates that onecould sample a field of onions using TIBS and thendetermine whether acceptable control would beachieved if the field were sprayed (assuming goodspray coverage).CONCLUSIONS and NEXT STEPS These studies indicatethat resistance to at least one insecticide, Warrior T,has occurred in some onion growing regions of NewYork, and that resistance appears to have developedbecause of practices in individual fields. Furthermore,susceptibility to Warrior in an individual field canchange dramatically within a single season. This pointsto the need to monitor fields routinely before choosingthe right insecticide. We are developing a TIBS systemto determine susceptibility to other classes ofinsecticides. If susceptibility to a particular class ofinsecticide can be determined quickly, as with TIBS,then it will be possible to develop an insecticideresistance management strategy for onion thrips ononions.REFERENCERueda, A, and A. M. Shelton. 2003. Development andevaluation of a thrips insecticide bioassay systemfor monitoring resistance in Thrips tabaci. PestMgt. Sci. (in press).A. M. Shelton, B. A. Nault, J. Plate & J. Z. ZhaoDepartment of EntomologyCornell University, NYSAESGeneva, New YorkUSAPyrethroid Susceptibility of Tobacco Budworm, Heliothis virescens (F.), and Bollworm, Helicoverpa zea (Boddie),in LouisianaABSTRACT Louisiana has maintained a statewidepyrethroid susceptibility monitoring survey for tobaccobudworm, Heliothis virescens (F.), and bollworm,Helicoverpa zea (Boddie), since 1986 and 1988,respectively. Adult males of both species werecaptured with pheromone baited wire cone traps andexposed to cypermethrin in an insecticide residual onglass (AVT) bioassay. During 1987 to 2002, annualsurvival of tobacco budworm adults exposed to 10µg/vial of cypermethrin using the AVT increased from15% to 60%. The percentage of parishes in whichgreater than or equal to 50% survival of tobaccobudworm was recorded increased from 8% in 1990 to>70% during 2002. Survival of bollworm adultsexposed to 5 µg/vial of cypermethrin in the AVTincreased from 2% in 1988 to 34% in 2002. Prior to1990, bollworm survival exceeded 11% in only oneparish. During 2002, survival exceeded 31% in six ofnine parishes. No control failures of bollworms treatedwith pyrethroids have been observed in Louisiana. In1995, pyrethroids no longer were recommended by theLouisiana Cooperative Extension Service for control oftobacco budworm.KEYWORDS Insecticide resistance, tobacco budworm,Heliothis virescens (F.), bollworm, Helicoverpa zea(Boddie), pyrethroids45

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2INTRODUCTION Insecticide resistance in key insectpests is an important issue for the cotton industry. Twoof the most important cotton pests in the mid-south andsoutheastern United States are the tobacco budworm,Heliothis virescens (F.), and the bollworm,Helicoverpa zea (Boddie). Resistance toorganochlorines, DDT, organophosphates (Sparks1981), and carbamates (Elzen et al. 1992) has beenreported with both species. Resistance in tobaccobudworm populations to pyrethroid insecticides, aswell as isolated field control failures were observed inArkansas (Plapp et al. 1987), Louisiana (Leonard et al.1987), Mississippi (Roush and Luttrell 1987), andTexas (Allen et al. 1987, Plapp et al. 1987) during1986. In response, pyrethroid resistance managementplans were implemented in those states to maintain theeffectiveness of pyrethroids for control of tobaccobudworm (Anonymous 1986). An importantcomponent of these plans was pyrethroid susceptibilitymonitoring for tobacco budworm. Resistance to DDTand organochlorine insecticides has been reported inbollworm (Sparks 1981). In 1998, field control failures(Walker et al. 1998) resulting from pyrethroid-resistantpopulations of bollworm (Brown et al. 1998) werereported in South Carolina. Monitoring of thesusceptibility of bollworm populations in Louisiana topyrethroids was initiated during 1988. To date no fieldcontrol failures of bollworm infestations treated withpyrethroids have been observed in Louisiana.MATERIALS and METHODS Adult vial bioassays (AVT)similar to those described by Plapp et al. (1987, 1990)were used to monitor the susceptibility of fieldcollected tobacco budworm and bollworm moths tocypermethrin. Stock solutions of cypermethrin weredeveloped by dissolving technical grade (98%)insecticide in acetone. Serial dilutions from each stocksolution yielded the desired concentrations. Theinterior surface of 20 ml glass scintillation vials wascoated with cypermethrin by pipetting 0.5 ml of theappropriate solution into vials. These vials were rotatedon a modified hot dog roller (heating elementdisconnected) until all of the acetone had evaporated.Vials were stored in a dark environment until used inbioassays.Male tobacco budworm and bollworm moths werecollected using wire cone traps (Hartstack et al. 1979)baited with synthetic sex pheromone lures (Hendrickset al. 1987) from May through September. Moths werecollected from parishes (tobacco budworm populationssurveyed in 2 to 17 parishes during 1986 to 2002,bollworm populations surveyed in 8 to 20 parishesduring 1988 to 2002) throughout the cotton productionregions of Louisiana (Figure 1). The insecticideconcentrations used in these surveys included10µg/vial cypermethrin for tobacco budworm and5µg/vial cypermethrin for bollworm. Moths wereplaced into insecticide-treated and control (non-treated)vials (one moth/vial) and mortality was determinedafter 24-h of exposure (HAE). Moths were considereddead if they were incapable of sustained flight for ca. 1minute. Data were corrected for control mortality usingAbbott's (1925) formula.RESULTS and DISCUSSION In response to field controlfailures of tobacco budworm with pyrethroids amonitoring program was initiated in Louisiana andcontinued through the present year (Graves et al 1994,Bagwell et al. 2001, Cook et al. 2003). Limited surveys(two parishes) of tobacco budworm susceptibility topyrethroids were conducted during 1986, andconcentrated on fields associated with unsatisfactorycontrol. Mean survival of tobacco budworm mothsfrom Bossier and Tensas parishes was 33% and 41%,respectively (Figure 2). In 1987, more extensivemonitoring efforts were conducted across Louisiana. In1988, only one parish had tobacco budworm survival>30% (East Carroll), while tobacco budworm survivalbetween 11% and 30% was observed in six parishes(Figure 3). During 1990, tobacco budworm survival inCatahoula parish was 51% (Figure 4). Tobaccobudworm survival between 31% and 50% wasobserved in seven parishes. During 1992, tobaccobudworm survival >50% was observed in two parishes(Figure 5). Survival in Madison Parish wasapproximately 60%. Tobacco budworm survivalbetween 31% and 50% was observed in ten parishes.Survival in excess of 50% was not observed in anyparish during 1994 (Figure 6). However, the number ofparishes in which survival between 31% and 50%increased. During 1996, tobacco budworm survivalbetween 31% and 50% was observed in 11 parishes(Figure 7). Survival >50% was observed in twoparishes with 66% tobacco budworm survival observed46

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2in East Carroll parish. During 1998, tobacco budwormsurvival >50% was observed in seven parishes, with78% survival recorded in Morehouse parish (Figure 8).Also, survival >30% was observed in eight parishes.Tobacco budworm survival >50% was observed inseven parishes during 2000 (Figure 9) representing ca.54% of the parishes in which monitoring wasconducted. Tobacco budworm survival >50% wasobserved in 86% of the parishes during 2002 (Figure10). In Catahoula parish during 2002, 83% survival oftobacco budworm adults exposed to cypermethrin inthe AVT was observed.Pyrethroid susceptibility surveys of Louisianabollworm were initiated during 1988. Bollwormsurvival was 10% was observed in three parishes of16 parishes during 1996 (Figure 15). During 1998,survival >10% was observed in 15 of 20 parishes withsurvival in one parish exceeding 30% (Figure 16).Bollworm survival >10% was recorded in nine parishesduring 2000 (Figure 17). During 2002, bollwormsurvival exceeded 10% in seven of nine parishes and infive parishes survival was >30%, with survival of 42%observed in one parish (Figure 18). Pyrethroidresistance in tobacco budworm gradually increasedfrom 15% statewide during the late 1980's to ca. 40%during the mid 1990's (Figure 19). Resistancemanagement (IRM) plans extended the useful life ofpyrethroids for tobacco budworm control until ca. 1995when they were removed for the Louisiana CooperativeExtension Service Recommendations for control oftobacco budworm in cotton, but they continued to beused for bollworm control. Alternative insecticides fortobacco budworm control became available in 1995, aswell as, Bollgard cotton varieties. During 1995 to 2002,tobacco budworm survival has steadily increased eventhough pyrethroids are not applied to control tobaccobudworm. This continued increase is probably theresult of inadvertent selection pressure from pyrethroidapplications targeting bollworm and other insect pestsof cotton.No field control failures of bollworm treated withpyrethroids have been observed in Louisiana.However, bollworm survival in the AVT has increasedsince 1996 (Figure 20). The highest annual survivalwas observed during the 2002 season (ca. 34%).Presently, pyrethroids are used to control bollworm innon-Bollgard and Bollgard cotton varieties. In addition,these products are also used against other cotton insectpests as well as bollworm in other crop hosts includingcorn, grain sorghum, and soybean.47

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Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2ACKNOWLEDGMENTS The assistance of LouisianaCounty Extension Agents, Louisiana AgriculturalExperiment Station personnel, and members of theLouisiana Agricultural Consultants Association duringthese surveys is appreciated. Also, the financial supportof the LSU AgCenter, Cotton Incorporated, andLouisiana's cotton producers is appreciated.REFERENCESAbbott, W. S. 1925. A method of computing the effectiveness of aninsecticide. J. Econ. Entomol. 18: 265-267.Allen, C. T., W. L. Multer, R. R. Minzenmayer, and J. S. Armstrong.1987. Development of pyrethroid resistance in Heliothispopulations in cotton in Texas, pp. 332-335. In Proceedings 1987Beltwide Cotton Conf., National Cotton Council, Memphis, TN.Anonymous. 1986. Cotton entomologists seek to delay pyrethroidresistance in insects. MAFES Res. Highlights 49:8.Bagwell, R. D., D. R. Cook, B. R. Leonard, S. Micinski, and E. Burris.2001. Status of insecticide resistance in tobacco budworm andbollworm in Louisiana during 2000, pp. 785-790. In Proceedings2001 Beltwide Cotton Conf., National Cotton Council, Memphis,TN.Brown, T. M., P. K. Bryson, D. S. Brickle, S. Pimprale, F. Arnette, M. E.Roof, J. T. Walker, and M. J. Sullivan. 1998. Pyrethroid-resistantHelicoverpa zea and transgenic cotton in South Carolina. CropProtection 17: 441-445.Cook, D. R., J. Temple, R. H. Gable, S. Micinski, W. F. Waltman, A. M.Stewart, B. R. Leonard, and R. D. Bagwell. 2003. Insecticidesusceptibility of Louisiana bollworm and tobacco budwormpopulations, (in press). In Proceedings 2003 Beltwide Cotton Conf.,National Cotton Council, Memphis, TN.Elzen, G. W., B. R. Leonard, J. B. Graves, E. Burris, and S. Micinski.1992. Resistance to pyrethroid, carbamate, and organophosphateinsecticides in field populations of tobacco budworm (Lepidoptera:Noctuidae) in 1990. J. Econ. Entomol. 85:2064-2072.Graves, J. B., B. R. Leonard, E. Burris, S. Micinski, S. H. Martin, C. A.White, and J. L. Baldwin. 1994. Status of insecticide resistance intobacco budworm and bollworm in Louisiana, pp. 769-774. InProceedings 1994 Beltwide Cotton Conf., National Cotton Council,Memphis, TN.Hartstack, A. W., J. A. Witz, and D. R. Buck. 1979. Moth traps for thetobacco budworm. J. Econ. Entomol. 75:519-522.Hendricks, D. E., T. N. Shaver, and J. L. Goodenough. 1987.Development of bioassay of molded polyvinyl chloride substratesfor dispensing tobacco budworm (Lepidoptera: Noctuidae) sexpheromone bait formulations. Environ. Entomol. 16:605-613.Leonard, B. R., J. B. Graves, T. C. Sparks, and A. M. Pavloff. 1987.Susceptibility of bollworm and tobacco budworm larvae topyrethroid and organophosphate insecticides, pp. 320-324. InProceedings 2000 Beltwide Cotton Conf., National Cotton Council,Memphis, TN.50

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Plapp, F. W., G. M. McWhorter, and W. H. Vance. 1987. Monitoring forpyrethroid resistance in the tobacco budworm in Texas-1986, pp.324-326. In Proceedings 1987 Beltwide Cotton Conf., NationalCotton Council, Memphis, TN.Plapp, F. W., Jr., J. A Jackman, C. Campanhola, R. E. Frisbie, J. B.Graves, R. G. Luttrell, W. F. Kitten, and M. Wall. 1990. Monitoringand management of pyrethroid resistance in tobacco budworm(Lepidoptera: Noctuidae) in Texas, Mississippi, Louisiana,Arkansas, and Oklahoma. J. Econ. Entomol. 83:335-341.Roush, R. T. and R. G. Luttrell. 1987. The phenotypic expression ofpyrethroid resistance in Heliothis and implications for resistancemanagement, pp. 220-224. In Proceedings 1987 Beltwide CottonConf., National Cotton Council, Memphis, TN.Sparks, T. C. 1981. Development of resistance in Heliothis (Helicoverpa)zea and Heliothis virescens in North America. Bull. Entomol. Soc.Am. 27: 186-192.Walker, J. T., M. J. Sullivan, S. Turnipseed, M. E. Roof, and T. M.Brown. 1998. Prospects for field management of pyrethroidresistantcorn earworm (cotton bollworm) populations in SouthCarolina, pp. 1145-1147. In Proceedings 1998 Beltwide CottonConf., National Cotton Council, Memphis, TN.D. R. Cook (Corresponding Author)LSU AgCenter, Dept. of Entomology402 Life Sciences Bldg.Baton Rouge, LA 70803USAPhone (225) 578-1839Fax (225) 578-1643Email dcook@agctr.lsu.eduB. R. LeonardLSU AgCenter, Dept. of EntomologyBaton Rouge, LA 70803R. D. BagwellLSU AgCenter, Scott Research and Extension CenterWinnsboro, LA 71295S. MicinskiLSU AgCenter, Red River Research StationBossier City, LA 71113J. B. Graves (Retired)LSU AgCenter, Dept. of EntomologyBaton Rouge, LA 70803Monitoring of Insecticide Resistance in Helicoverpa armigera (Hübner) from 1998 to 2002 in Côte d'Ivoire, WestAfricaHelicoverpa armigera (Hübner) is the major insectpest of the cotton crop in West Africa. Populationsrecently developed resistance to pyrethroids via theoverproduction of oxidases. To control this pest, aresistance management strategy was applied in themajor West African cotton growing countries from1999 onwards. In Côte d'Ivoire insecticide resistance ofH. armigera was monitored in field strains from 1998to 2002 using vial tests and topical applications inthird-instar larvae. Vial tests with discriminating dosesof cypermethrin were used directly on field-collectedlarvae at the end of the cotton season. The percentageof resistant larvae varied around 67% with 6 µg/vialand around 13% with 30 µg/vial according to year andplace.Topical applications were used in the laboratoryon the first generation of populations collected fromcotton (Gossypium hirsutum), tomato (Lycopersicumesculentum), or a strongly infested ornamental flower(Antirrhinum majus) with various insecticides. Theresistance factors calculated from dose-mortalityregressions varied from 5 to 38 with deltamethrin.They were always higher for strains collected fromcotton in the Bouaké area at the end of the season.Concerning the pyrethroid alternatives currently usedin Côte d'Ivoire, no lack of susceptibility was detectedwith endosulfan and profenofos in the cotton fieldstrains showing their interest in the resistancemanagement strategy. All these results suggest arelative stability of the pyrethroid resistance in H.armigera for 1998 to 2002 and confirm the success ofthe resistance management strategy.KEY WORDS Helicoverpa armigera, insecticideresistance, cotton.INTRODUCTION More than two million small-scalefarmers are cultivating cotton in West Africa on anaverage of 1 ha plots. Cotton is one of the mostimportant cash crops in the region and provides morethan 50% of the cash to the agricultural populations. Itcontributes largely to the struggle against poverty inthe countries of the African Cotton Belt. Cotton cropsare damaged by a large number of insect species, themost harmful being the cotton bollworm Helicoverpaarmigera (Hübner). To control the whole cotton pestcomplex, available and profitable approaches includechemicals associated with the use of hairy cultivar andappropriate cultural practices. As a result, this cropreceives the largest amount of insecticide among thecrops cultivated in the area. Since the early eighties,pyrethroids have been extensively used, because theyare very efficient against bollworms at low doses andbecause their toxicity to mammals is low. However,since 1996 high infestations of H. armigera wereobserved in some areas suggesting control failure dueto the development of insecticide resistance (Vassal etal., 1997; Vaissayre et al., 1998; Martin et al., 2000). In1998, pest infestations extended to several countries inWest Africa despite an increase in spaying intensity.This event became highly threatening since similarresistance observed in India and Pakistan resulted indramatic losses of cotton production, pushing somefarmers to suicide (McCaffery et al., 1988 and 1989).Facing this problem, West African countries put51

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2together efforts to better understand the phenomenon,studying the resistance mechanism. It appears thatresistance was due to greater degradation ofpyrethroids in resistant insects involving oxidases fromthe cytochrome P450 family (Martin et al., 2002). Anetwork group, named PR-PRAO (Prevention andManagement of Pyrethroid Resistance in H. armigera)was implemented, involving CIRAD, IRAC, and all theactors of cotton protection in West Africa fromresearch to extension services and advisory services toinitiate management strategies and monitor programsto survey the resistance level of insecticides used.The control of H. armigera on the West Africancotton regions has been the focus of an insecticideresistance management strategy (IRM) restricting theuse of pyrethroids in cotton (Ochou et al., 1998, Ochouand Martin, 2002). This strategy has been generallysuccessful in all West African cotton-growing regionssince 1999. As no difference in oxidases activity hasbeen described for resistance to endosulfan, weconcluded that pyrethroid resistance would not cross.As this insecticide already proved to be very efficientagainst the bollworm before the introduction ofpyrethroids, endosulfan was chosen to replacepyrethroids in the beginning of the cotton season.In order to assure a sustainable resistancemanagement strategy, it was essential to survey thepyrethroid resistance levels in each cotton-growingregion because of the migrating habits of H. armigera(Nibouche, 1994). In the present investigation wemonitored the pyrethroid resistance level in H.armigera from 1998 to 2002 in the cotton-growing areaof Côte d'Ivoire. Populations were collected in cotton(Gossypium hirsutum), in tomato (Lycopersicumesculentum), and in a strongly infested ornamentalflower (Antirrhinum majus). The susceptibility ofpyrethroid alternatives such as endosulfan andprofenofos has also been surveyed.EXPERIMENTAL PROCEDUREInsects. A susceptible H. armigera strain (BK77) wasoriginally collected in Côte d'Ivoire in 1977 and rearedin CIRAD Entomological Laboratory fromMontpellier, France. Larvae were reared on artificialdiet at 25°C, 75% humidity and at 12h/12h photoperiodin the laboratory as previously described (Couilloudand Giret, 1980).Field samples of different stages of H. armigerawere collected from 1998 to 2002. Samples wereobtained from strongly infested crops in identifiedfarmers' fields from the cotton-growing area. Thestrains were named according to the nearest large town(BK: Bouaké; SAR: Sarhala; OGL: Ouangolo; NIO:Niofoin; MKN: Mankono; BOU: Boundiali) with thecollection date (year/month) and the crop name: 'c' forcotton, 't' for tomato, 'g' for gumbo, and 'f' for theornamental flower. A minimum of 50 larvae werecollected in each field and reared in the laboratory ofthe Centre National de Recherche Agronomique(CNRA) in Bouaké on an artificial diet for onegeneration at 25°C. The adults were placed in cagesand fed on a 5% honey solution. Their eggs werecollected on sterilised gauze and washed with 1%bleach.Insecticides. The insecticides used were all technicalgrade materials. Deltamethrin (99%) and endosulfan(99%) were obtained from Aventis CropScience,France. Cypermethrin (93.2%) was obtained fromFMC, USA. Profenofos (95%) was provided bySyngenta, Abidjan, Côte d'Ivoire.Topical application. Standard third-instar larvae topicalbioassays were used to determine insecticide toxicity.Five serially diluted concentrations were prepared. Foreach concentration, 10 third-instar larvae (35-45 mg)were treated with 1 µl of solution applied bymicroapplicator to the thorax. Each test was replicated3 times and included acetone treated controls.Mortality in the controls was less than 10%. Afterdosage, the test larvae were held individually at 25°Cand 75% humidity. Mortality was assessed 72h aftertreatment. Larvae were considered dead if unable tomove in a co-coordinated way when prodded with aneedle. LD50 was determined by using the Finneymethod (1961). Transformations and regression lineswere automatically calculated by DL50 1.1 software ofCIRAD.Vial tests. Vials were impregnated with technicalcypermethrin in acetone. Two discriminating doseswere chosen: 5 µg/vial which killed 100% of thesusceptible larvae from BK77 strain, and 30 µg/vialwhich killed 100% of the susceptible larvae and 60 to80% of a resistant population collected in Benin in1997 (Vaissayre et al., 2002). The tubes were kept indarkness at ambient temperature. Extension serviceagents conducted vial tests for four years in Octoberduring the strong infestation at the end of the cottonseason. The three first years they worked in the areas ofBouaflé, Bouaké, Boundiali, Ferké, or Tortiya. In 2001,vial tests were conducted in twelve areas spread overthe Center, West North, and North of the country.Larvae of H. armigera measuring 1 to 1.5 cm werecollected from farmer cotton fields at least seven daysafter the last treatment. Two replications wereconducted in different location. Each larva was placedin a vial without any food. The vials were kepthorizontal and protected from heat. Larval mortalitywas assessed at 24 h. Larvae were considered dead ifunable to move in a coordinated manner.52

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2RESULTSVial tests. The vial test method wasdirectly used in cotton field of farmers toconfirm and survey the pyrethroidresistance of H. armigera in the wholeWest African cotton-growing region.Because of low infestations since theapplication of the IRM strategy for 1999,vial tests could be used only at the end ofthe cotton season. The results obtained inCôte d'Ivoire with two discriminatingdoses of cypermethrin are illustrated inFigure 1.The first discriminating dose (6µg/vial) showed high percentages ofresisting larvae varying from 40 to 90%.With the second discriminating dose (30µg/vial) the percentage of resisting larvaevaried from 1 to 35%. On average, therewas less than 20% of resisting larvae with30 µg/vial of cypermethrin; this thresholdcorresponding with control failures in field(Vaissayre et al., 2002). Moreover this vialtest method applied for the same period inMali, Burkina Faso, Benin, and Togo,showed that H. armigera populations fromCôte d'Ivoire have lower percentages ofresisting larvae.Despite of the low level of infestationduring the survey, these results showed:1. the presence of pyrethroid resistinglarvae in all populations tested and2. high variations between thepopulations tested.Bioassays. In the same period, bioassays with topicalapplications were used to give results on the annualevolution of the pyrethroid resistance level in fieldpopulations. Dose-mortality regression lines were donewith deltamethrin in the first generation of fieldpopulations collected from different locations from1998 to 2001. LD50s varied from 0.30 to 1.05 µg/gindicating a low resistance level (maximum RF=20)(Fig.2).To know the seasonal evolution of the pyrethroidresistance level in a location, dose-mortality regressionlines were done in the first generation of all H.armigera populations collected each year from varioushost plants in Bouaké area. Data showed thatdeltamethrin LD50s in H. armigera varied from 0.4 to2 µg/g (Fig. 3). Therefore, the resistance factor (RF)for field populations varied from 10 to 38 foldcompared to the susceptible strain BK77. It was verydifficult to find H. armigera in vegetables because ofthe sparse small plots and the high number oftreatments. The ornamental flower A. majus, cultivatedwithout any treatment in small plots near the CottonResearch Station of Bouaké, appeared to be veryattractive for H. armigera and could be very useful inthe future to collect populations throughout the dryseason. The highest resistance level was annuallyobserved in populations collected from cotton inOctober corresponding to the last period of treatments.The resistance level slightly decreased in the dryseason as shown by the resistance level of thepopulations collected from ornamental flowers. Thisresult was observed as well in H. armigera populationscollected in Benin (Djinto et al., to be published)suggesting a fitness cost of the pyrethroid resistance.H. armigera populations collected in October fromcotton in Bouaké seemed to be always the moreresistant among field populations. This result can beexplained by the high number of treatments in varietymultiplication plots of the Cotton Research Station ofBouaké. Interestingly, the deltamethrin toxicity of53

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2of cross-resistance with pyrethroids(Martin et al., 2002). It is the same for theprofenofos that did not show any resistancein H. armigera field populations (Fig. 6).This organophosphorus compound wasused since 1977 in mixtures withpyrethroids to control the mites. It wasused alone for 2000 as pyrethroidsalternative. No cross-resistance wasdetected with pyrethroids (Martin et al.,2002).these populations has been surveyed in each year since1985 (Fig. 4). Three plateaux appeared showingschematically the apparition of pyrethroid resistance inH. armigera populations from Bouaké: susceptibility(1985-1988), decrease of susceptibility (1989-1995)and resistance (1996-2001).Concerning the survey of pyrethroid alternatives,dose-mortality regression lines for endosulfan andprofenofos have been done annually since 1999 inBouaké populations. Endosulfan LD50s of fieldpopulations were at the same level than BK77 (Fig. 5).Thus endosulfan did not show any resistance in H.armigera cotton field populations. This cyclodiene isused from the 1970's in mixtures with DDT andmethyl-parathion. It was replaced by pyrethroids in1984. But from the development of pyrethroidresistance, endosulfan was reused alone with success(Ochou and Martin, 2002). From this result, it appearsthat this success partially originates from the absenceDISCUSSION Results obtained on larvaewith the application of discriminatingdoses by vial test method confirmed thepresence of resistant H. armigera in all thefield populations collected in Côte d'Ivoire.The larva vial test was not an accuratemethod. However, results obtained directlyin the field were indicators of thepyrethroid resistance and could beconfirmed with the bioassay method.Bioassays results showed that thedeltamethrin resistance level in H.armigera populations could be consideredas low in Côte d'Ivoire (in average RF

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2ACKNOWLEDGEMENTS The authors thankthe research and development unit team ofcotton companies (CIDT, IC, and LCCI)and the technical assistance of T. Konate,I. Ouattara, and M.J. Mousso of theentomological laboratory of the CNRA(Centre National de RechercheAgronomique).REFERENCESCouilloud, R. and Giret, M. (1980). Multiplicationd'Helicoverpa armigera Hübner: améliorationspossibles grâce à l'adoption d'une techniqued'élevage en groupe de chenilles. Coton et FibresTropicales, 35: 217-224.Finney, D. J. (1971). Probit analysis. 3rd Edition,Cambridge University Press pp.333.Forrester, N.W., Cahill, M., Bird, L. J. and Layland, J. K.1993 Management of pyrethroid and endosulfanresistance in Helicoverpa armigera (Lepidoptera:Noctuidae) in Australia. In Bull. Entomol. Res.Supplement Series N°1. International Institute ofEntomology pp. 1-132.Gunning, R. V. and Easton, C. S. (1993) Resistance toorganophosphate insecticides in Helicoverpaarmigera (Hübner) (Lepidoptera: Noctuidae) inAustralia. Gen. Appl. Entomol., 25: 27-34.McCaffery, A. R., Maruf, G. M., Walker, A. J. andStyles, K. (1988). Resistance to pyrethroids inHeliothis spp.: Bioassay methods and incidence inpopulations from India and Asia. Brighton CropProtection Conference - pests and diseases -, 433-438.McCaffery, A. R., King, A. B. S., Walker, A. J. and El-Nayir, H. (1989). Resistance to syntheticpyrethroids in the bollworm Heliothis armigerafrom Andhra Pradesh, India. Pesticide Science, 27:65-76.McCaffery, A. R. (1998). Resistance to insecticides inheliothine Lepidoptera. Phil. Trans. R. Soc. Lond. B pp. 1735-1750.Martin, T., Ochou, O. G, Hala N'Klo, F, Vassal and J. M., Vaissayre, M.(2000). Pyrethroid resistance in the cotton bollworm, Helicoverpaarmigera (Hübner), in West Africa. Pest Management Science, 56:549-554.Martin, T., Chandre, F., Ochou, O. G., Vaissayre, M. and Fournier, D.(2002). Pyrethroid resistance mechanisms in the cotton bollwormHelicoverpa armigera (Lepidoptera: Noctuidae) from West Africa.Pesticide Biochemical Physiology, 74 : 17-26.Martin, T., Ochou, O. G., Vaissayre, M. and Fournier D. (2002). Positiveand Negative Cross-resistance to Pyrethroids in Helicoverpaarmigera from West Africa. Resistance Pest ManagementNewsletter, 12, 1.Nibouche, S. (1994). Cycle évolutif de Helicoverpa armigera (Hübner)dans l'ouest du Burkina Faso. Thèse de Doctorat, 15/12/1994. EcoleNationale Supérieure Agronomique, Montpellier, France. pp.143.Ochou, O. G., Martin, T. and Hala N'Klo, F. (1998). Cotton insect pestproblems and management strategies in Côte d'Ivoire, W. Africa.Proceedings of the World Proceedings of the World CottonResearch Conference-2. Athens, Greece, September 6-12, pp. 833-838.Ochou, G. O. and Martin, T. (2002). Pyrethroïd Resistance inHelicoverpa armigera (Hübner): Recent Developments andProspects for its Management in Côte d'Ivoire, West Africa.Resistance Pest Management Newsletter, 12, 1.Vaissayre, M., Vassal, J. M. and Martin, T. (1998). Pyrethroid resistancein the bollworm Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) in West Africa. Proceedings of the World CottonResearch Conference-2. Athens, Greece, September 6-12, pp. 701-705.Vaissayre, M., Vassal, J. M., Staetz, C. and Irving, S. (2002). A Vial TestMethod for the Survey of Pyrethroid Resistance in Helicoverpaarmigera in West Africa. Resistance Pest Management Newsletter,12, 1.Vassal, J. M., Vaissayre, M. and Martin, T. (1997). Decrease in thesusceptibility of Helicoverpa armigera (Hübner) (Lepidoptera:Noctuidae) to pyrethroid insecticides in Côte d'Ivoire. ResistancePest Management 9, 2, 14-15.T. MartinCNRA, Programme cotonBP 633, BouakéCôte d'Ivoire&CIRAD-CA, Programme coton, TA72/0934398, Montpellier cedex 5FranceG. O. OchouCNRA, Programme cotonBP 633, BouakéCôte d'IvoireM. VaissayreCIRAD-CA, Programme coton, TA72/0934398, Montpellier cedex 5FranceD.FournierIPBS-UMR 5089205 Route de NarbonneF-31077 ToulouseFrance55

Susceptibility Level of Colorado Potato Beetle (Leptinotarsa decemlineata Say) to some Pyrethroids andNereistoxin Derivative (Bensultap) Insecticides in Poland in 2002INTRODUCTION Poland is a major producer of potatoes(Solanum tuberosum). This plant is cultivated on about1,000,000 ha (2002) in this country but the averagecrop is very low - 17.2 t/ha. Colorado potato beetle(CPB) (Leptinotarsa decemlineata Say) is the mostserious potato pest in Poland and considered to be thePolish pest with the highest likelihood of developinginsecticide resistance (Wegorek et al. 2001) All classesof insecticide have been widely used to control CPB inPoland. A 50 year period of intensive selectionpressure has lead to CPB resistance to five majorchemical classes of insecticide: chlorinatedhydrocarbons, organophosphates, carbamates,nereistoxin analoques, and pyrethroids (Pruszynski etal. 1988, Wegorek et al. 2001).For many years pyrethroids (deltamethrin,cypermethrin, alpha-cypermethrin, zeta-cypermethrin,lambda cyhalothrin, esfenvalerate, fenvalerate, andethofenprox) and bensultap have held the primary placein CPB control in Poland. Over a period of 26 years forpyrethroids and 17 years for bensultap, these twoclasses of insecticides have been most commonly usedfor controlling CPB in Poland (Szczesna et al. 1990,Przybysz et al. 1996, Pawinska M. 2000, MrowczynskiM. 2000). Nowadays the use of pyrethroids and thenereistoxin derivative (bensultap) is systematicallydecreasing. The cases of observed CPB resistance forboth classes under practical conditions have increased.Bioassays of determined pyrethroids andnereistoxin derivative (bensultap) for resistancemonitoring in CPB were performed in western, central,and northern regions of Poland (Institute of PlantProtection in Poznan, Plant Breeding andAcclimatization Institute in Bonin, and Institute ofOrganic Industry in Warsaw). Project P06r 126 21 issupported by the Polish State Committee For ScientificResearch.METHODSLaboratory Tests: In laboratory tests the standardmethod recommended by Insecticide Resistance ActionCommittee (IRAC method nr.7) was used. Fourteeninsect populations were used from three regions ofPoland: 5 populations from the western region - WinnaGora, Rogalinek, Plewiska, Skoki, and Bolewice; 3populations from northern region - Czarnoszyce,Bonin, and Zamarte; and 6 populations from the centralregion - Bielawy, Rudka, Radachowek, Gorzno,Chobot, and Rabiez. A representative sample of CPBlarvae (2-st-instar) in selected field populations andsufficient non-infested, untreated leaves were collectedfor testing.Chemicals:Pyrethroids -alpha-cypermethrin (commerciallyavailableproduct Fastac 100 EC) 20, 10, 5ppm concentrations were tested(recommended concentration in Poland: 20-33 ppm)lambda-cyhalothrin (commerciallyavailableproduct Karate 025 EC ) 10, 5, 2,5ppm concentrations were tested(recommended concentration in Poland: 15-25 ppm)deltamethrin (commercially-availableproduct Decis 2,5 EC ) 20, 10, 5 ppmconcentrations were tested (recommendedconcentration in Poland: 15-25 ppm)Nereistoxin analogues -bensultap (commercially-available productBancol 50 WP ) 20, 10 ppm concentrationswere tested (recommended concentration inPoland: 500- 667 ppm)Accurate dilutions of the tested compound fromcommercially available products were used indetermined doses.Leaves were dipped in water for untreated controland other leaves in tested insecticide concentrationliquids for about five seconds and placed on papertowel to dry. Untreated and treated dry leaves wereplaced into 10 cm diameter Petri dishes with 10 cmdiameter filter paper and 10 larvae were placed in eachdish. 3-5 replicates were conducted for eachconcentration and control.A final assessment of the lethal effects of thepyrethroides insecticides were determined after 72hours and assessment of the nereistoixin analogue(bensultap) was determined after 120 hours afterapplication and expressed as percent mortality at eachdose, correcting for untreated (control) mortalitiesusing Abbott's formula (Abbot 1925). Untreatedmortalities were quoted.At each assessment, larvae were classed as either:(a) unaffected, giving a normal response (such astaking a co-ordinated step) when gently stimulated bytouch, or (b) dead or affected, the latter giving anabnormal response to stimulation. Corrected Mortality= 100 x (P-C/100-C) where P = % mortality intreatment, C = % mortality in controls.

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Tests were performed in the laboratory withconditions of 22-24 degrees C and a photoperiod of16:8 (L;D).RESULTS and DISCUSSIONPopulations from all three provinces demonstratedsome level of resistance to one or more pyrethroidsinsecticides (deltamethrin, alpha-cypermethrin,lambda-cyhalothrin). In laboratory studies (2002) thepyrethroids insecticides were less effective incontrolling CPB larvae. Survival at 20ppmconcentration in the case of alpha-cypermethrine anddeltamethrin and 10ppm concentration in the case oflambda-cyhalothrin indicated the occurence of strongfield resistance in tested populations.The populations from the central and northernregions of Poland were more resistant to testedpyrethroids than populations from the western region.CPB strains tested in 2002 were not tolerant tonereistoxin analogue (bensultap). The LC50 data forbensultap from 1998 (western populations) rangedfrom 11.86 to 14.59 (Wegorek et al. 1999) were notsignificantly different than data in 2002. The resultsindicated that populations tolerant for pyrethroids werenot cross-resistant to bensultap. The widespread use ofpyrethroids in Poland can lead to control failure.Understanding the conditions that favour thedevelopment, the causes, and the mechanisms ofresistance are the crucial challenges for the future ofpyrethroid use to CPB control in Poland.The constant monitoring of CPB susceptibilitylevels to insecticides used in Poland and studies onmechanisms of CPB resistance to those insectivideswill allow for the development of the best strategy fordelaying CPB resistance. At present the generalprinciples of the management strategy involve therational application of all recommended insecticides57

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2and their rotation includingdifferent modes of theirtoxic action (Wegorek etal. 1998).To conserve as long aspossible the highinsecticidal potency of allchemical classes ofinsecticides in Poland it isnecessery to follow thegeneral resistancemanagement guidelines,which were elaborated bythe Institute of PlantProtection in Poznan withhelp of IRAC (Wegorek etal. 2002). These guidelinescould be adopted in allareas of potato insecticidalprotection in Poland. Forthis reason simple fieldtests are recommended.The InsecticideSusceptibility TestMethod for CPBResistance DetectionPlant protectionadvisors and farmers inPoland should considerusing the simple IRACmethod nr.7 before "highrisk"CPB populationtreatment to detect the fieldefficacy of insecticides forCPB control.1. Collect arepresentative sample(300 - 400) of CPBlarvae L2 (or L3) stagein the different placesof the field.2. Collect sufficient noninfested,untreatedleaves to perform thetest.3. Wearing solvent-proofgloves, syringe orpipette and prepareaccuraterecommended (field concentration) water dilutionsfrom commercially available products.4. Dip leaves in water for untreated control and otherleaves in the tested liquid for 5 seconds and placeon paper towel to dry.5. Place the untreated and treated dry leaves incontainers, which must be suitable for keepingenough leaf material in good condition for up 2 -3days.6. Add equal numbers of L2 (or L3) CPB larvae toeach container (one container should be use for58

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2untreated control) but no more than 20larvae/container and store the containers in thearea where they are not exposed to direct sunlightor extremes temperature (a mean temperature of22-240 C is preferred).7. For rapidly acting insecticides (pyrethroids,chloronicotinyls, phenylpyrazoles,organophosphores, and carbamates) a finalassessment of larval mortalities is made after 48 h.For slowly acting insecticides (bensultap, Bacillusthuringiensis, etc.) assess after 120 h.Larval mortality in control container should be lessthan 10%. Larval mortality in treatment should to be100%. If 1 or more larvae survive the treatment test theproduct should not be recommended to control thetested population.REFERENCES:1. Abbot W. S. 1925. A method of computing the effectivnessof aninsecticide. J. Econ. Ent. 18: 265-267.2. Pawinska M., Mrowczynski M. (2000) Occurence and control ofColorado potato beetle in 1978-1999 (summary inEnglish).Wystepowanie i zwalczanie stonki ziemniaczanejLeptinotarsa decemlineata Say w latach 1978-1999. [W:] Progressin Plant Protection 40 (1), 292-293. Pawinska M. (2000) Colorado Potato Beetle with emphasis onproblems in Central and Eastern Europe. [W:] Proceeding of thefourh World Potato Congress Amsterdam,The Netherlands 4-6 September 2000,Wagenningen Pers, 195-2024. Pruszynski S., Wegorek P., KroczynskiJ., Szczesna E. Zwalczanie stonkiziemniaczanej w sezonie 1988. OchronaRoslin 6, 4-6. 1988.5. Przybysz E., Pawinska M., Wegorek P.1996. Resistance monitoring studies ofCPB on some insecticides applied inPoland (summary in English). Monitoringodpornosci stonki ziemniaczanej naniektore stosowane w Polsce insektycydy.Progress in Plant Protection / Postepy wOchronie Roslin Vol. 36, No. 1: 338-343.6. Szczesna E., Wnuk S., Kroczynski J.,Wegorek P. 1990 Skutecznosc preparatuEnolofos w swietle badan laboratoryjnychoraz doswiadczen przeprowadzonychprzez niektore WSKiOR w 1989 r. Mat.XXX Sesji Nauk. IOR, I 181-188, 1990.(summary in English)7. Wegorek P., Pawinska M., PrzybyszE.1999 Monitoring odpornosci stonkiziemniaczanej na chlorfenwinfos, cypermetryne i bensultap.Progress in Plant Prot./Postepy w Ochr. Rosl. Vol.39. 1. 351-359.(summary in English)8. Wegorek P., Mrowczynski M., Wachowiak H. 2001 Courrent status ofresistance in Colorado Potato beetle (leptinotarsa decemlineata Sayin Poland. Mat Conf. Resistance 2001 Meeting the Challange.Rothamsted 46 p.9. Wegorek P., Mrowczynski M., Dutton R., Pawinska M., Przybysz E.2002. Insecticide resistance management strategy for Coloradopotato betle (leptinotarsa decemlineata Say.) in Poland. PlantProtection Inst. in Poznan. 16 pp.Dr. Pawel WegorekInstitute of Plant ProtectionPoznanPolandProf. Dr. Hab. Stefan PruszynskiInstitute of Plant ProtectionPoznanPolandDr. Maria PawinskaPlant Breeding and Acclimatization InstituteDepartment in BoninPolandMgr. Anna PrzybyszInstitute of Organic IndustryWarsawPolandSusceptibility Level of Colorado Potato Beetle (Leptinotarsa decemlineata Say) to Phenylpyrazole andChloronicotinyl Insecticides in Poland in 2002INTRODUCTION Insecticides from the phenylpyrazoleand chloronicotinyl (neonicotinoid) classes arerelatively new for the control of CPB Poland (fipronil -1996, acetamiprid - 1996, imidacloprid - 1998,thiamethoxam - 1999, thiacloprid 2002) (Pawinska etal. 1995,1996, Mrowczynski et al.1997, Wegorek et al.2001). The importance of both new classes ofinsecticide for CPB control in Poland will besystematically increased and it may be assumed thatthe CPB resistance will occur in these new classes too.The use of bensultap, the inhibitor of theacetylocholine receptor, the target of chloronicotinylinsecticides (Yamamoto et al.1995) was widespread inPoland during 17 last years.The constant monitoring of CPB susceptibilitylevel to these classes of insecticides in Poland and59

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2studies on mechanisms of CPB resistance to them willallow the development of an enterprising strategy fordelaying CPB resistance. Bioassays of phenylpyrazolesand chloronicotinyls for resistance monitoring in CPBhave been performed at the Institute of Plant Protectionin Poznan since 2001(Wegorek et al. 2001)MATERIALS and METHODSLaboratory Tests Method: In laboratory tests thestandard method recommended by the InsecticideResistance Action Committee (IRAC method nr.7) wasused. A representative sample of CPB insects (larvae 2-st-instar or second generation adults) in selected fieldpopulations and on sufficient non-infested, untreatedleaves was collected for testing.CPB Populations: The 6 insect populationsoriginated from three regions of Poland (Winna Gora,Rogalinek, Skoki, and Bolewice from western Poland,Rarwino from northern Poland, and Badrzychowicefrom central Poland).Accurate dilutions of the tested compound fromcommercially available product were used indetermined doses.Chemicals:Chloronicotinylsimidacloprid (commercially-available productConfidor 200 Sl) LC50 and LC 95 werecalculated (recommended field concentration30-50 ppm)acetamiprid (commercially-available productStonkat 160 SL) LC50 and LC 95 werecalculated (recommended field concentration30-50 ppm) )thiamethoxam (commercially-availableproduct Actara 25 WG) ) LC50 and LC 95were calculated (recommended fieldconcentration 50-67 ppm)Phenylpyrazolesfipronil (commercially-available productRegent 200 S.C.) LC50 and LC 95 werecalculated (recommended field concentration50- 67 ppm)Leaves were dipped in water for untreated controland other leaves in tested insecticides liquid for aboutfive seconds and placed on paper towel to dry.Untreated and treated dry leaves were placed into 10cm diameter Petri dishes with 10 cm diameter filterpaper, and 10 larvae or adults insects were placed ineach dish. 5 or 10 replicates were conducted for eachconcentration and control.A final assessment of the lethal effects of thephenylpyrazoles and chloronicotinyls insecticide wasdetermined 72 hours after application and expressed aspercent mortality at each dose, correcting for untreated(control) mortalities using Abbott's formula (Abbot1925). Untreated mortality was quoted. The lethalconcentration at which > 50% of insects are killed -LC50 (ppm) and lethal concentration at which > 95% ofinsects are killed -LC 95 (ppm) were calculated usingthe probit analysis Finney method (Finney 1952).At each assessment, larvae or beetles were classedas either (a) unaffected, giving a normal response (suchas taking a co-ordinated step) when gently stimulatedby touch, or (b) dead or affected, the latter giving anabnormal response to stimulation or showing abnormalgrowth which should be described. Corrected Mortality= 100 x (P-C/100-C) where P = % mortality intreatment, C = % mortality in controls.Tests were performed in the laboratory withconditions of 22-24 degrees C and a photoperiod of16:8 (L;D).RESULTS and DISCUSSION Laboratory investigations gaveno indication of resistance elicited by testedpopulations of CPB to insecticides from phenylpirazoleand chloronicotinyl groups (fpronil, thiamethoxam,imidacloprid, and acetamiprid). No significantdifferences in LC50 and LC95 values and no evidence60

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2of cross-resistance to pyrethroid insecticides weredetected. The LC50 and LC95 values were very similarwith those from 2001 (Wegorek et al. 2001), disclosingno increased tendency for any population to toleratehigher concentrations of the tested insecticides. Thebioassay method reported is considered well suited formonitoring the response of CPB to phenylpirazole andchloronicotinyl insecticides. Since in one growingseason CPB usually produces only one completegeneration, and on average 1.5 applications are used onit, there is small risk of a fast development of highresistance to a given biologically activeingredient or a chemical group over a fewseasons. However, a continual selectionpressure with similar products must beavoided, and insecticides from differentchemical classes and with differentmechanisms of action should be used.Using phenylpirazoles andchloronicotinyls in Poland we must rememberthat CPB is multiply and cross-resistant to fivemajor groups of insecticides in United States(Forgash 1985), inclding resistance to Bacillusthuringiensis (Whalon 1997), abamectine(Argentine, Clark 1990), and imidacloprid(Grafius and Bishop 1995)) and tolerance tofipronil (Colliot et all 1992).REFERENCES:1. Abbot W. S. 1925. A method of computing theeffectivnessof an insecticide. J. Econ. Ent. 18: 265-267.2. Argentine J. A., and Clarc J. M. 1990. Selection forabamectin resistance in Colorado potato betle(Coleoptera: Chrysomelidae). Pestic. Sci. 28:17 - 24.3. Argentine J. A., Clarc J. M., Lin H. 1992. Genetics andBiochemical Mechanisms of Abamectin Resistance inTwo Isogenic Strains of Colorado Potato Beetle.Pesticide Biochemistry and Phisiology 44, 191-207.4. Colliot F. Kukorowski K. A., Hawkins D. W., Roberts D. A.1992. Fipronil: a new soil and foliar broad spectruminsecticude. Ibid. 1: 29 - 34.5. Finney D. J. 1952. Probit analysis (A statistical treatment ofthe sigmoid response curve) Cambridge Univ. Press:236-245.6. Forgash, A. J. 1985. Insecticide resistance in Coloradopotato beetle. Pp. 33-52 in Proceedings of thesymposium on the Colorado potato beetle, XVIIthInternational Congress of Entomology, ed.D.N. Ferroand R. H. Voss. Res. Bull. 704, Mass. Agric. Expt. Sta.,Amherst, Mass.7. Grafius E. J., Bishop B. A. 1996. Resistance to imidaclopridin Colorado potato beetle in Michigan. Department ofEntomology Michigan State University8. Mrowczynski M., Wachowiak H., Pawinska M. 2000.Wyniki badan wdrozeniowych insektycydu Actara 25WG w zwalczaniu stonki ziemniaczanej (Leptinotarsadecemlineata Say.). Progress in Plant Protection. Postepyw Ochronie Roslin, 40, 2: 903-906. (summary inEnglish)9. Pawinska M, Turska E. 1995. Zastosowanie imidaklopridu wochronie plantacji ziemniaka. Materialy XXXV SesjiNaukowej Instytutu Ochrony Roslin w Poznaniu. Czesc I- Referaty: 296 - 302. (summary in English)10. Pawinska M., Mrówczynski M. Wachowiak H. WiderskiK., Bubniewicz P., Glazek M. Jablczynski W. 1996.61

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Studies on application of fipronil (Regent) in the control of pests onagricultural crops (summary in English) Badania nadzastosowaniem Fipronilu w zwalczaniu szkodników uprawrolniczych. 1996 Progress in Plant Protection/ Postepy w OchronieRoslin Vol. 36 (2) 108-112.11. Wegorek, M. Mrówczynski, H. Wachowiak. 2002. "Chloronicotinoidsand phenylopirazoles insecticides - susceptibility level and activityagainst Colorado potato beetle (Leptinotarsa decemlineata Say) inPoland". Potato Research 2002, vol 44, issue 3, p 307 .12. Whalon M. E., Utami Rahardja, Pathara Verakalasa 1997. Selectionand management of Bacillus thuringiensis-resistant Colorado potatobeetle. Advances in Potato Pest Biology and Management : 309-325.13. Yamamoto I., Yabuta G., Tomizawa M., Saito T. Miyamoto T.Kagabu S. 1995 molecular mechanism for selective toxicity ofnicotinoids and neonicotinoids. J. Pesticide Sci. 20, 33 - 40.Dr Pawel WegorekInstitute of Plant ProtectionPoznanPolandRelative Resistance in Open and Greenhouse Populations of Scirtothrips dorsalis Hood (Thysanoptera:Thripidae) on Rose to Dimethoate and AcephateABSTRACT LC50 values for two commonly usedinsecticides viz., dimethoate and acephate in open fieldand greenhouse populations of Scirtothrips dorsalisHood on rose collected from Bangalore, India werecalculated. The LC50 values varied from 0.1072 -0.0253 % in the case of dimethoate and 0.0309 -0.1455 % in the case of acephate. Greenhousepopulations of S. dorsalis have developed 1.5 and 4.7fold resistance to dimethoate and acephate respectivelyin comparison to open field populations.KEY WORDS: Scirtothrips dorsalis, insecticideresistance, roseINTRODUCTION The chilli thrips, Scirtothrips dorsalisHood (Thysanoptera: Thripidae) is one of the mostdevastating pests of several agricultural and62

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2horticultural crops worldwide. Roses are being grownboth under open and greenhouse conditions and S.dorsalis is a major pest of this crop, especially undergreenhouse conditions. Dimethoate and acephate, apartfrom the new compounds ethofenprox andimpadacloprid, are recommended for control of thispest (Jhansi Rani and Eswara Reddy, 2001; Nair et al.,1991). Growers have repeatedly observed thatdimethoate and acephate have not given satisfactorycontrol against this pest in and around Bangalore,particularly under greenhouse conditions. Reddy et al.(1992) have documented the relative resistance inthrips, S. dorsalis, to different conventional insecticideson chilli from Andhra Pradesh in India. However,information regarding relative susceptibility of S.dorsalis with regard to open and greenhousepopulations from India is not available. Therefore, thepresent trial was taken up with the objective todetermine the relative susceptibility of two populationsof S. dorsalis on rose, from open fields andgreenhouses, to dimethoate and acephate.MATERIALS and METHODS Populations of S. dorsaliswere collected both from the field and fromgreenhouses from the Indian Institute of HorticulturalResearch (IIHR), Bangalore. Two insecticides viz.,dimethoate 30 EC (Rogor) and acephate 75 SP(Starthane) as commercial formulations were used forthe study. The assay procedure followed was modifiedfrom Reddy et al (1992). Tender rose leaves weredipped in suspensions of different concentrations foreach insecticide, air dried for 30 minutes, andintroduced into glass vials. Using a fine brush, tennymphs from homogeneous populations of S. dorsaliswere carefully transferred to each concentration ofinsecticide treated leaves (open and greenhousepopulations in two separate sets). The vials were sealedwith parafilm and minute holes were made forventilation. Leaves dipped in water alone were used ascontrol. Each concentration of insecticide treatmentand control were replicated thrice. Mortality countswere taken 24 hours after the release of the test insects.Based on the number of insects that responded to thedifferent concentrations of insecticides, a probitanalysis was carried out for arriving at LC50 values forboth open field and greenhouse populations using acomputer aided MSTATC package. The resistanceindex (RI) was computed according to the formulasuggested by FAO (1979) as, RI = LC50 of resistantstrain/LC50 of susceptible strain.RESULTS and DISCUSSION Data on the LC50 values ofthe two insecticides between two populations of thrips,S. dorsalis, revealed that LC50 values varied from0.1072 - 0.0253 % in the case of dimethoate and from0.0309 - 0.1455 % in the case of acephate (Table 1).Greenhouse populations were less susceptible whencompared to field populations in cases where bothinsecticides were tested. When the resistance index wastaken into consideration, greenhouse populations of S.dorsalis had developed 1.5 and 4.7 fold resistance todimethoate and acephate, respectively when comparedto open field populations (Table 1). The differentialresponse of thrips to the two insecticides in the study interms of LC50 values was attributed to thedevelopment of resistance by S. dorsalis in greenhousepopulations when compared to open field populations.Relatively less susceptibility of S. dorsalis populationsin greenhouses may be attributed to their frequentexposure to different insecticides (nearly at fortnightlyintervals) and the quick elimination of susceptiblepopulations/genes in comparison to open populations.However, in the case of open populations, there isrelatively more chance of passing of susceptible genesin successive generations by mating with resistantpopulations, resulting in more susceptible populationswhen compared to greenhouse populations.Earlier reports have indicated that citrus thrips,Scirtothrips citri (Moulton) developed resistance inDDT, dimethoate, acephate, bendiocarb, andformentanate (Morse and Brawner, 1986 and Immarajuet al., 1989). S. dorsalis, a dominant species of thripson roses, may have undergone selection for a numberof insecticides in the past which might have led tocross-resistance to related compounds that are widelyused. Hence, there is an urgent need to curbindiscriminate insecticide use on roses particularly ingreenhouses. The result also suggests that futurecontrol programmes for S. dorsalis on rose ingreenhouses need to incorporate a resistancemanagement strategy as a major component.ACKNOWLEDGEMENT The authors are grateful to theDirector, Indian Institute of Horticultural Research,Bangalore for providing necessary facilities and Dr.N.K.Krishna Kumar, Head of Division of Entomologyand Nematology for suggesting the study.REFERENCES:FAO, 1979. Recommended methods for the detection and measurementof resistance for adult aphids. FAO Method no.17 FAO PlantProtection Bulletin 27: 29-32.Immaraju, J.A., Morse, J.G. and Kersten, D.J. 1989. Citrus thrips(Thysanoptera:Thripidae) Pesticide resistance in Coachella and SanJoaquin valley of California. Journal of Economic Entomology 82:374-380.Jhansi Rani, B. and Eswara Reddy, S.G. 2001. Efficacy of selectedinsecticides against thrips, Scirtothrips dorsalis Hood on rose in63

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2greenhouse. Pest Management in Horticultural Ecosystems. 7 (1):54-58.Morse, J.G. and Brawner,O.L. 1986. Toxicity of pesticides of Scirtothripscitri and implications to resistance management. Journal ofEconomic Entomology. 79:560 570.Nair, V.V., Regunath and Visalakshi, A. 1991. Control of thripsScirtothrips dorsalis Hood on rose. Entomon, 16 (4): 327-329.Reddy, G.P.V., Deva Prasad, V. and Srinivasa Rao, R. 1992. Relativeresistance in chilli thrips, Scirtothrips dorsalis Hood populations inAndhra Pradesh to some conventional insecticides. Indian J. PlantProt. 20(2): 218 - 222.V. Sridhar and B. Jhansi RaniDivision of Entomology and NematologyIndian Institute of Horticultural ResearchHessaraghatta Lake Post, Bangalore-560 089IndiaStatus of Pyrethroid Resistance in Helicoverpa armigera in IndiaHelicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) is charismatic and one of the most dreadedinsect pests in agriculture, accounting for theconsumption of over 30% of the total insecticide useworldwide. The frequent and rapid changes in croppingpatterns and agro-ecosystems, the polyphagus nature ofthe pest, and its cosmopolitan abundance haveaccentuated the problem globally. The problems of thispest are magnified due to its direct attack on fruitingstructures, its voracious feeding habits, its highmobility and fecundity, its multivoltine, overlappinggenerations with facultative diapause, its nocturnalbehaviour, migration, and host selection by learning,and a propensity for acquiring resistance againstinsecticides (Satpute and Sarode, 1995; Sarode, 1999).This pest has been recorded feeding on 182 plantspecies across 47 families in the Indian subcontinent,of which 56 are heavily damaged and 126 are rarelyaffected (Pawar et al., 1986). Losses due solely to thispest of up to Rs.10,000 million have been reported incrops like cotton, pigeonpea, chickpea, groundnut,sorghum, pearl millet, tomato, and other crops ofeconomic importance (Raheja, 1996).In India, Helicoverpa is represented by threespecies, with H. armigera constituting 99.2%, H.peltigera at 0.6%, and H. assulta at 0.2% of the totalpopulation (Pawar, 1998). In recent years, H. armigerahas assumed such serious proportions in the countrythat for the past decade, farmers and plant protectionagencies of central and state governments havevirtually become perplexed regarding its control whichultimately has lead to an array of social, economical,and political problems. Of these, a primary problemconcerns the development of resistance in this pest to anumber of insecticides including pyrethroids.Resistance to pyrethroids in H. armigera had beenreported from a number of countries through out theworld including India. The control failures of syntheticpyrethroids were first detected on pigeonpea against H.armigera at Lam farm, Guntur, A.P. in 1986. From1987-88 to 1989-90, continuous monitoring andevaluation of the H. armigera population revealed thatthe resistance levels were low during 1988-89 inAndhra Pradesh, decreased by a factor of 10 (Table 1).During the cotton season 1989-90, it increased nearly2-fold greater than that encountered in 1988-89. It mayalso be elucidated that the resistance response inNorthern India, i.e. the Delhi and Karnal strains,remained constant. It was also shown that theresistance was not restricted to one or the otherpyrethroid, but had extended to all the three pyrethroidsused in the country viz., cypermethrin, fenvelerate, anddeltamethrin (Mehrotra, 1991). The subsequent studiesconfirmed the major cause of crop failure in A.P. wasresistance to synthetic pyrethroids in this pest(Srivastava, 1995). Although, the pyrethroid resistancein H. armigera was found to be restricted to anapproximately 75km wide and 200km long beltcomprising three districts of A.P. viz. Prakasham,Guntur, and parts of Krishna (Dhingra et al., 1988), thepresence of increased tolerance to cypermethrin in thetwo strains collected in Tamil Nadu from cotton (1989)and groundnut (1991) suggested that resistance topyrethroids was wide spread and could be featuredthroughout South India (Armes et al., 1992). InCoimbatore, resistance to cypermethrin was found to64

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2be 25- to140-fold during 1992 and 1993 (Armes et al.,1996), but despite a reduction in the use of pyrethroidsin the state over the past few years, resistance levelsincreased 64- to 207-fold (Kranthi et al., 2001).The pyrethroid resistance in H. armigera hassubstantially increased in certain regions of Central andNorthren India too. Although, the pyrethroid use in thedistricts Akola and Amravati of Central India is not ashigh as in the Warangal or Guntur districts of A.P., thehighest level of pyrethroid resistance was recorded inthese districts during the H. armigera outbreak of1997-1998. This is in sharp contrast to resistance levelsreported from the Bhatinda district of Punjab wherepyrethroid use was reasonably high (Kranthi et al.,2001). In the Varanasi area in Uttar Pradesh, pyrethroidresistance was recorded in H. armigera larvae collectedfrom early pigeonpea in November 1991 and fromchickpea in March 1992 (Armes et al., 1992). Thesedetails reveal that the pyrethroid resistance has alreadymoved from South India to other parts of India.A survey of insecticide resistance in H. armigeraon the Indian sub-continent during 1991-95 revealedthat pyrethroid resistance levels were highest in theintensive cotton and pulse growing regions of Centraland Southern India where excessive application ofinsecticides is common (Armes, 1996). However, it hasbeen observed that several regions of the countrywhere insecticides are used in a very low quantity,resistance in this pest can be expected over space andtime (Tripathy and Singh, 1999). Among the severalpossibilities in this regard, many workers suspected itto be the resultant of immigration of resistant moths ina windward direction either from the North Indianstates of Punjab and Haryana where to control this pest,pyrethroid use was ever increasing (Pedegley et al.,1987) or from Central India (Vaishampayan and Singh,1995).Thus, the possibility of dispersal or migration ofH. armigera, that may occur at particular times duringor after cropping seasons eventually influence theresistance patterns across the country. Hence, revisedinsecticide resistance management (IRM) strategies areurgently required if further widespread failures tocontrol this pest are to be avoided.REFERENCES:Armes, N. J., Jadhav, D. R., Bond, G.S. and King, B.S. 1992. InsecticideResistance in Helicoverpa armigera in South India. PesticideScience 34:355-364.Armes, N. J., Jadhav, D.R. and DeSouza, K.R. 1996. A survey ofInsecticide resistance in Helicoverpa armigera in Indian subcontinent.Bulletin of Entomological Research. 86: 499-514.Dhingra, S., Phokela, A. and Mehrotra, K.N. 1988. Cypermethrinresistance in the population of Heliothis armigera Hubner. NationalAcademy of Science Letters, 11: 123-125.Kranthi, R.K., Jadhav, D., Wanjari, R., Kranthi, S. and Russell, D. 2001.Pyrethroid resistance and mechanism of resistance in field strains ofHelicoverpa armigera (Lepidoptera: Noctuidae). Journal ofEconomic Entomology .94(1): 253-263.Mehrotra, K.N. 1991. Current status of pesticide resistance in insect pestsin India. Journal of Insect Science. 4:1-4.Pawar, C.S. 1998. Helicoverpa- A national problem which needs anational policy and commitment for its management. Pestology.12(7): 51-59.Pawar, C.S., Bhatnagar, V.S. and Jadhav, D.R. 1986. Heliothis speciesand their natural enemies with their potential for biological control.Proceedings of Indian Academy of Sciences (Animal Sciences). 95:697-703.Pedegley, D.E., Tucker, M.R. and Pawar, C.S. 1987. Wind born migrationof Heliothis armigera (Hubner) (Lepidoptera: Noctuidae) in India.Insect Science and its applications. 8: 599-604.Raheja, A.K. 1996. IPM Research and Development in India: Progressand Priorities. In : Recent Advances in Indian Entomology (Ed.O.P.Lal). APC Publications Pvt. Ltd., New Delhi, pp.115-126.Sarode, S.V. 1999. Sustainable management of Helicoverpa armigera(Hubner). Pestology. Spl. Issue 13(2):279-284.Satpute, U.S. and Sarode, S.V. 1995. Management of Heliothis on cotton-A thought. In: Souvenir published at the State Level Conference onIPM. May 26, 1995 Akola (Maharashtra). pp.27-31.Srivastava, C.P. 1995. Insecticide resistance in Helicoverpa armigera inIndia. Resistant Pest Management. 7(1).Tripathy, M.K. and Singh, H.N. 1999. Circumstantial evidences formigration of resistant moths of Helicoverpa armigera at Varanasi.Uttar Pradesh. Indian Journal of Entomology. 61(4): 384-395.Vaishampayan, S.M.Jr. and Singh, H.N. 1995. Evidence on migratorynature of Heliothis armigera (Hubner) adults collected on light trapsat Varanasi. Indian Journal of Entomology. 57(3): 224-232.R.K.Arora, M.Yaqoob, and Arvind IsharDivision of EntomologyFaculty of Agriculture, SKUAST-JammuUdheywalla Jammu -180 002IndiaIntegrated Resistant Management of Codling Moth Cydia pomonella L. in ItalyINTRODUCTION Trentino and Emilia-Romagna are twoof the major apple-growing regions in Italy, with ayearly apple production of 450,000 and 210,000 tonnesa year, respectively. Emilia-Romagna is also the mostimportant area for pear production, with 630,000tonnes of pears grown annually. The codling moth(Cydia pomonella) is the key pest affecting Italianapple and pear orchards; those pest populations haveuntil now been primarily managed by insect growthregulators (IGRs) and organo-phosphate applications(OP). Subsequent to the increase in the damage causedby the codling moth at the end of the 1990s, amonitoring programme was started in 1998 in order todetect resistance, and field trials were carried out inorder to define the most appropriate IRM strategies(Ioriatti and Bouvier, 2000).MONITORING of the RESISTANCE Resistance monitoringwas carried out by using two methods: the attracticidesusceptibility test was used to evaluate the azinphosmethylactivity on the feral male moths, whileresistance to the IGR diflubenzuron was evaluated bytreating the over-wintering larvae collected directly inthe field (Ioriatti et al., 2003).65

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2According to the results, codling moth resistancewas spread differently across the two regions:in Emilia-Romagna half of the samplescollected, when exposed to the resistancemonitoringtest for azinphos methyl anddiflubenzuron, showed a significant reduction inmortality in comparison to the susceptible strain;in Trentino the situation was less serious: thesamples analysed proved to be less susceptiblethan the reference strain, although the differencewas not statistically significant. A significantdecrease in mortality was only detected in asmall area.Recently, the apple-dipping test has been used todetect resistance directly on the larvae collected fromthe infested fruits in Trentino and Emilia-Romagna.According to the dose-response line evaluated fordifferent insecticides by Charmillot (in press) on asusceptible laboratory strain, a discriminating dose waschosen for diflubenzuron, flufenoxuron, indoxacarb,and spinosad. The collected larvae were divided intofour classes according to weight. The results obtainedhave shown that the mortality caused by thediscriminating concentration was closely linked to theweight of the field-collected larvae, even in thesusceptible strain. Only the first class of larvae, lessthan 10 mg in weight, responded to the treatment, aswas expected from the previously used monitoringtests. The preliminary results confirmed that there wasa large difference in mortality between susceptible andresistant (i.e.Copparo) strains when treated withdiflubenzuron, while the tested codling moth strainsshowed only a slight reduction when treated with theother three insecticides (Fig.1).damage within acceptable limits, and on the other,defining new strategies based on less toxic controlmeasures.IGR: As a result of their environmentally friendlycharacteristics, insect growth regulators are still themost suitable insecticides for Integrated FruitProduction, and their use for the codling moth controlis preferable when they prove to be effective. The fieldefficacy of IGR insecticides has been evaluated inthese two fruit-growing regions. In Emilia-Romagnathe trials were performed in orchards that had over thepast few years recorded a rise in damage in spite of theincrease in the number of insecticide treatments. On theother hand, in Trentino, where the level of the CMpopulation is generally lower, the tests were carried outon fruit-farms where CM proved to be less susceptiblethan the reference strain, although the difference wasnot statistically significant.Emilia-Romagna: The efficacy of two IGRs hasbeen tested (Boselli et al, 2001) in a pear-growingfruit-farm with a population of resistant CM. Twotreatments of flufenoxuron were applied against thefirst generation of carpocapsa, in comparision with twodiflubenzuron treatments. The first treatment for eachof the products was applied at the beginning of egglaying.For both products the second application wasrepeated 15 days after the start of the treatment. Undera severe infestation (27% of the fruits attacked in theuntreated plots) diflubenzuron cannot ensure anadequate crop protection (48.1% efficacy), whileflufenoxuron achieved a good efficacy rate (94.4%efficacy) (Fig.2). These results confirm thatflufenoxuron loses its ovicidal effect when used againsta resistant population, while maintaining its efficacyupon the larvae (Sauphanor et al., 1998).EFFICACY of DIFFERENT INSECTICIDES upon SENSITIVE andRESISTANT POPULATIONS of CYDIA POMONELLA Duringthe monitoring on the territory of the resistant CMpopulations, a series of field tests have been planned inthe two regions with a view to assessing, on the onehand, which active substances were capable ofconstituting a valid alternative for the containment ofTrentino: Several IGRs (flufenoxuron,diflubenzuron, novaluron, lufenuron) were comparedon an apple orchard against the first generation of CM.The first generation lasts longer than in the southernregions and three insecticide applications were neededto cover the entire egg-laying period. At the end of thefirst generation fruit damage in the untreated plot was66

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.235%. The efficacy of the 4 IGRs did not differ greatlyand ranged from 90 to 95% (Fig.3).Chlorpyriphos: It is used in Emilia-Romagna andTrentino for pest-control in orchards affected by a highCM density and where resistance to either azinphos-mor diflubenzuron was detected. The choice was madeaccording to the results that demonstrated no crossresistancewith azinphos methyl. As a matter of fact,the attracticide susceptibility test carried out on malemoths showed that there was not a reduction inmortality when applied on the azinphos-resistant strainof codling moth (Fig. 4).beneficial organisms, new strategies have beenevaluated based on less toxic control measures with aview to replacing chlorpyrifos in the IRM.In Emilia-Romagna, field trials were conducted ina pear orchard where the previous year's harvest fruitdamage had been 85%, in spite of the application of asmany as 12 treatments. The efficacy of differentinsecticides was assessed against the first generation ofcarpocapsa (Boselli et al, 2001). All the treatmentswere based on one application of flufenoxuron, at thestart of egg-laying, followed by two larvacidetreatments with different active ingredients(carpovirusine, chlorpyriphos, chlorfenapyr,methoxyfenozide, indoxacarb, tebufenozide, andspinosad) against the new-born larvae. The results atthe end of the first generation show that there were nosignificant differences between six of the seveninsecticides compared. In this case there is a suspicionthat the flufenoxuron applied at the start of the seasonhad actually contributed to significantly limiting thefinal damage in all the experimental regimes, thusreducing the differences between the various productsbeing tested (Fig. 5). Only the fruit damage in thechlorfenapyr plots resulted significantly higher than theIn Emilia-Romagna field tests were again carriedout on the first generation. Such tests comprised thetwo applications of chlorpyriphos targeted to the newbornlarvae, preceded by an action with different IGRs(diflubenzuron, flufenoxuron, esaflumuronteflubenzuron, methoxyfenozide, triflumuron,lufenuron) applied at the beginning of the egg-layingperiod. As a further treatment two applications ofchlorpyriphos were applied. All the treatments werestatistically differentiated from the untreated plot, butnot among themselves. Indeed, just two applications ofchlorpyrifos provided the same efficacy as thosestrategies where an IGR was applied at the beginningof the season.The EVALUATION of NEW STRATEGIES BASED on LESSTOXIC CONTROL ACTIONS As chlorpyriphos does nothave a good profile in terms of toxicity for humans andothers.New field trials were organised in orchards wherethe presence of resistant CM populations had beendemonstrated, in order to evaluate the efficacy of theinsecticides when used alone, with no IGR as the initialtreatment. The products used were the granulosis virusand new products like indoxacarb, spinosad, andthiacloprid; azinphos-methyl was used as a standard ofreference. The treatments were applied at the start ofegg hatching and repeated eight days later (Boselli etal, 2001).67

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2The results support the excellent local larvicideactivity of the virus-based products, spinosad andthiacloprid, whose efficacy was superior, even if not ina statistically significant way, to azinphos-methyl.Instead, the average damage recorded in the plotstreated by indoxacarb was greater than with thecompared insecticides, thus not ensuring sufficientprotection (10.7% damage) (Fig. 6).In Trentino the field experiments were carried outin different years and with different CM populationdensities. The insecticides evaluated were spinosad,methoxyfenozide, thiacloprid, and indoxacarb ascompared with chlorpyriphos. Each experimentalregime comprised a treatment with an IGR at the startof egg-laying. Different insecticides were appliedaccording to the intensity of egg-laying recorded byscouting in the orchard. Only chlorpirifos was appliedcuratively, 3 times in high-pressure years and 2 timesin low-pressure years. In this context, spinosad andthiacloprid resulted to be as effective asmethoxyfenozide and indoxacarb (Fig. 7).insecticide resistance was first detected. The firstobjective to be achieved in the farms with high fruitdamages was to reduce such damage within acceptablelevels. Hence, plant protection strategies weredeveloped that provided for integration betweenbiological and chemical products. As regards the IGRs,diflubenzuron was substituted by flufenoxuron, whichshowed it could maintain a good degree of efficacy inpopulations resistant to this class of product. The use offlufenoxuron on pear orchards, however, is notadvisable because of its secondary effects on psillapopulations.Chlorpyriphos, because of its demonstrated lack ofcross-resistance with the azinphos-resistant strain, wascritical in managing resistance in both regions. The useof chlorpyriphos has to be limited due to its hightoxicity for humans and its negative side effects onbeneficial organisms. The reduction of organophosphatesuse has for the moment been made possibleby the use of the granulosis virus that demonstrates anefficacy equal to or superior to chemical products.Hence, it is now being widely employed in Emilia-Romagna. Moreover, in this region CM-GV and theapplication of mating disruption are the main strategiessuggested for orchards with low CM population levelsfor the resistance management.In Trentino, on the other hand, as the levels of theCM population are generally quite low, the wideapplication of mating disruption allowed pesticideresistance to be successfully managed. Furthermore,according to the results of the field trials carried out inthese two fruit-growing areas, it has been shown thatnew pesticides, such as spinosad and thiacloprid, couldbe introduced into the IRM programs. Nevertheless,attention should be paid to their possible negative sideeffectsagainst the beneficial organisms controlling thepear-psilla populations.REFERENCESBoselli M., Vergnani S.(2001). Attività di alcuni insetticidi nei confrontidella prima generazione di Cydia pomonella L. Informatorefitopatologico. 6: 40-46.Ioriatti C., Bouvier J.C. (2000). La resistenza agli insetticidi; il caso dellacarpocapsa (Cydia pomonella L.). Informatore Fitopatologico 9: 5-10.Ioriatti C., Bouvier J.C., Butturini A., Cornale R., Tiso R. (2003).Carpocapsa: la situazione della resistenza ad azinphos methyl ediflubenzuron in Trentino ed Emilia-Romagna. InformatoreFitopatologico. 1: 53-60.Sauphanor B., Brosse V., Monier C., Bouvier J.C. (1998). Differentialovicidal and larvicidal resistance to benzoylureas in the codlingmoth, Cydia pomonella. Entomol. exp. appl., 88: 247-253.C.IoriattiISMAAS.Michele a/A TrentoItalyCONCLUSIONS Technical recommendation IRMstrategies were applied in both regions after someM.BoselliServizio FitosanitarioRegione Emilia-Romagna, BolognaItaly68

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2A.ButturiniServizio FitosanitarioRegione Emilia-Romagna, BolognaItalyR.CornaleCentro Agricoltura AmbienteCrevalcore (Bologna)ItalyS.VergnaniCentro Ricerche Produzioni VegetaliCesenaItalyFungicide ResistanceResistance to QoI Fungicides in Podosphaera xanthii Associated with Reduced Control of Cucurbit PowderyMildew in Research Fields in the Eastern United StatesINTRODUCTION Application of fungicides continues tobe the principal practice for managing powderymildew, the most common disease of cucurbit cropsthroughout the world. Powdery mildew, which iscaused by Podosphaera xanthii, needs to be controlledon both leaf surfaces to avoid premature death ofleaves. It is especially important to control powderymildew on the underside of leaves where conditions aremore favorable for disease development than on uppersurfaces. Using systemic or translaminar fungicides isthe best approach. Unfortunately, most of thesefungicides are at risk for resistance developmentbecause they have single-site modes of action. Thecucurbit powdery mildew fungus has demonstrated ahigh potential for developing resistance. Each chemicalclass active for powdery mildew that is at risk forresistance somewhere in the world following repeateduse has developed resistance. Presence of resistantstrains has been associated with control failure. Thusmanaging fungicide resistance is an important aspect ofeffectively managing powdery mildew (McGrath2001).The fungicide program that has beenrecommended recently in the United States is astrobilurin fungicide (azoxystrobin formulated asQuadris® or trifloxystrobin formulated as Flint®)applied in alternation with the DMI fungicidemyclobutanil (formulated as Nova® or Rally®) tankmixedwith a protectant fungicide. This program usestwo strategies for managing resistance:1. alternation among systemic fungicides in at leasttwo chemical classes, and2. inclusion of protectant fungicides which are not atrisk for resistance development because they havemulti-site modes of action.Strobilurins are in fungicide group 11, the quinoneoutside inhibitor (QoI) activity group. Myclobutanil isa triazole fungicide in the DMI activity group, which isfungicide group 3. Quadris and Nova have beenavailable for commercial use for powdery mildew oncucurbits in the US beginning in 1998 when theyreceived Section 18 registration in some states. USfederal (Section 3) registration was granted for Quadrisin March 1999 and Nova in May 2000. Flint wasregistered in September 1999.Resistance is a major concern with QoI fungicidesused for cucurbit powdery mildew due to past historywith this pathogen developing resistance. Therefore itis important to monitor fungicide efficacy and, if poorperformance occurs, to determine if it is due toresistance. These were the goals of this study. Efficacywas monitored in NY. Pathogen isolates were alsoassayed for resistance from other areas where poorcontrol was reported.MATERIALS and METHODS Quadris applied in alternationwith Nova tank-mixed with chlorothalonil (formulatedas Bravo Ultrex®) was included in fungicide efficacyexperiments conducted with pumpkin from 1998 to2002 in Riverhead, NY. Quadris was used alone on aweekly schedule for another treatment in 2002 becauseother fungicides used with Quadris in a programdesigned for managing resistance, as recommended forproduction fields, might provide enough control ofpowdery mildew to mask the presence of strobilurinresistant strains, especially if they were at a lowfrequency. Fungicides were applied weekly with atractor-mounted boom sprayer. Applications wereinitiated after the IPM threshold of one leaf withsymptoms of 50 old leaves examined was reached in all(or almost all) plots (McGrath, 1996b). A randomizedcomplete block design with four replications was used.Upper and lower surfaces of 5 to 50 leaves, dependingon incidence, in each plot were examined weekly forpowdery mildew. Average severity for the entirecanopy was calculated from the individual leafassessments. Area under disease progress curve(AUDPC) values were calculated as a summationmeasurement of powdery mildew severity over thetreatment period. Cultural practices used and otherfungicide treatments tested are described in previousreports (McGrath 2000, 2002b; McGrath and Shishkoff1999, 2001, 2003). One other treatment was Flintapplied alone weekly in 1998 to obtain manufacturerrequestedefficacy data.An additional opportunity to evaluate Quadrisapplied alone or with Nova plus Bravo was provided69

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2through an experiment conducted with muskmelon in1999 to evaluate resistance management strategies forDMI fungicides. A preliminary report has beenpublished (McGrath and Shishkoff 2000).Fungicide sensitivity was determined for isolatesobtained in 2000 from a fungicide efficacy experimentconducted in research fields in Freeville, NY, wherecontrol obtained with pyraclostrobin formulated asBAS 500 was not as effective as expected based onprevious results (Zitter et al 2001). Isolates were alsocollected from the efficacy experiment conducted inRiverhead, NY, for comparison. A leaf-disk bioassaywith 0, 0,2, 2, 20, and 40µg/ml pyraclostrobin was usedto determine strobilurin sensitivity (McGrath et al1996). Assays were repeated at least once for eachisolate. Fungicide sensitivity was determined forisolates obtained from commercial fields in CA andfungicide efficacy experiments conducted in researchfields in AZ, CA, GA and NC, as well as NY, wherecontrol obtained with strobilurin fungicides was noteffective in 2002, contrasting with previous years.Leaves were collected on 22 July, 8 Oct, and 17 Octafter the last of four, five, and six applications of astrobilurin fungicide (Flint or Quadris) made inexperiments conducted by J. David Moore in Chula,GA, M. T. McGrath in Riverhead, NY, and Gerald J.Holmes in Clayton, NC, respectively. Leaves were alsocollected from non-treated (control) plants and plantsthat had been treated weekly with triadimefonformulated as Bayleton® in GA. Isolates were obtainedfrom the leaves. Strobilurin sensitivity was determinedusing 0, 0.5, 5, 50, and 100µg/mltrifloxystrobin. Sensitivity totriazole fungicides was alsodetermined using 5, 50, and100µg/ml triadimefon.2002). Reduced control in 2002 was evident for thetreatment with Quadris alternated with Nova and Bravoas well as when Quadris was used alone (Table 1).Reduced efficacy was also observed in KY, NJ, IL, MI,and VA in 2002 (W. Nesmith, S. A. Johnston, M.Babadoost, M Hausbeck, and C. Waldenmaier,personal communications).Isolates of Podosphaera xanthii resistant tostrobilurin fungicides were obtained from the GA, NC,and NY research fields. Four of nine NY isolates, 19 of21 GA isolates, and 13 of 15 NC isolates from plantstreated weekly with Flint or Quadris were able to growwell on leaf disks treated with 100 µg/mltrifloxystrobin in the bioassay. Strobilurin resistancewas also detected in the research fields in VA (Olaya,personal communication). Strobilurin sensitivityappeared to be qualitative as reported in other areas ofthe world (Ishii et. al. 2001). The maximumconcentration tolerated by most of the 73 isolates fromGA, NC, and NY in 2002 (89%) was either 0.5 or 100µg/ml trifloxystrobin. Azoxystrobin baseline sensitivitydistribution had been investigated in North America. Inone study with P. xanthii isolates collected in 1998 and1999 from several locations in North America, thegeometric mean of the baseline was 0.258 µg/ml andthe individual values ranged from 0.107 to 0.465 µg/ml(Olaya et. al 2000). In another study, 0.5-1 µg/ml wasthe maximum concentration tolerated by 60% of 72isolates collected from 1990 to 1996 in six states; 6%were able to grow, but only slightly, on leaf diskstreated with 5 µg/ml (Shishkoff and McGrath,RESULTS and DISCUSSIONStrobilurins used alone on aweekly schedule (use pattern notlabeled) did not effectivelycontrol cucurbit powdery mildewin 2002 in several fungicideefficacy experiments conductedin research fields. Isolates ofPodosphaera xanthii were collected for testingfrom fields in CA, GA, NC, and NY. Powderymildew had been controlled well by Flint orQuadris applied alone and Quadris applied inalternation with Nova and Bravo in fungicideefficacy experiments conducted yearly from1998 to 2001 in NY (Tables 1 and 2). Flintused alone effectively controlled cucurbitpowdery mildew in efficacy experimentsconducted in other states before 2002. Degreeof control in 2001 was 100% in GA (Langston et al2002), 80% in MI (Hausbeck et al 2002), 91% in VA(Alexander et al 2002), and 90% in DE (Everts et al,70

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2unpublished). This indicates that poor control withstrobilurins under field conditions was associated withreduced sensitivity in vitro. The resistant isolates wereable to tolerate at least 100-fold higher concentration ofstrobilurins than isolates with baseline sensitivity.Two strobilurin sensitive isolates and threeresistant isolates collected in 2002 responded similarlywhen tested in another laboratory using kresoximmethyland pyraclostrobin (H. Ypema, personalcommunication). These findings and experiences inother areas of the world with strobilurin-resistant P.xanthii indicate that cross-resistance probably extendsamong multiple strobilurins (Ishii et. al. 2001).All 14 isolates obtained from non-treated plants inthe GA experiment were sensitive to trifloxystrobin(maximum concentration tolerated was 0 mg/ml for14% of the isolates, 0.5 µg/ml for 79%, and 5 µg/ml for7%). Thus applying Flint alone shifted the pathogenpopulation substantially. Large changes in thefrequency of resistance in P. xanthii populations duringa growing season have been detected with triadimefonand benomyl (McGrath 1996a).Results on resistance in the western US areinconclusive as only 1 isolate could be tested from AZand only 5 from CA because spores obtained fromthese leaves grew poorly in culture. However, the AZisolate tolerated 50µg/ml trifloxystrobin. Also, one CAisolate grew on disks treated with 50 and 100 µg/mltrifloxystrobin in one assay, but growth was reducedcompared to lower concentrations. It did not survive tobe re-tested. The maximum concentration tolerated bythe other 4 CA isolates was 0.5µg/ml trifloxystrobin.Efficacy of strobilurin fungicides changed substantiallyfrom 2001 to 2002 in the CA research field (T. Turini2002, 2003). Control of powdery mildew achieved onlower leaf surfaces with the strobilurin fungicidesCabrio (pyraclostrobin), Quadris, and Flint used alonewas 65% - 94% in 2001 whereas severity did not differsignificantly from non-treated plants in 2002. Incontrast, the DMI fungicides Rally and Procure(triflumizole) used alone provided 82% - 100% controlin 2001 and 82% - 98% control in 2002. Strobilurinfungicides used alone in AZ provided 46% - 77%control of powdery mildew on lower leaf surfaces in2002 while triflumizole provided 91% control(Matheron et.al. 2003).Strobilurin resistance did not appear to be a factorin pyraclostrobin efficacy being lower in 2000(76%)(Zitter et al 2001) than in 1999 (92%)(Drennanet al 2000) in Freeville, NY. The highest concentrationtolerated by P. xanthii isolates obtained from theFreeville experiment in 2000 was only 2µg/mlpyraclostrobin. This concentration was tolerated by80% of Freeville isolates and 83% of isolates testedfrom Riverhead, NY, in 2000.The strobilurin-resistant isolates also exhibitedreduced sensitivity to DMI fungicides. All isolates butone tolerated 100µg/ml triadimefon. The one isolateunable to grow on leaf disks treated with 100 µg/mlwas able to tolerate 50µg/ml triadimefon. Resistance toDMI fungicides is quantitative. Isolates able to tolerate100µg/ml triadimefon are resistant to triadimefon butsensitive to myclobutanil because in fungicide efficacyexperiments Bayleton was ineffective while Nova waseffective where these isolates occurred (McGrath et. al.1996). Applying the DMI fungicide Bayleton weeklyin the experiment conducted in GA also shifted thepathogen population to a high frequency (71%) ofisolates that were resistant to strobilurin fungicides andinsensitive to DMI fungicides. Using strobilurinfungicides was shown in 1999 to be effective formanaging DMI resistance (McGrath and Shishkoff2000). In 2002, however, it appears that mostindividuals in the powdery mildew fungal populationthat were insensitive to one of these chemical classeswere also insensitive to the other, consequentlyapplying either a strobilurin or a DMI fungicide shiftedthe population towards insensitivity to both. None ofthe 73 isolates tested were strobilurin-resistant andDMI-sensitive; 7% were strobilurin-sensitive and DMIinsensitive.Only 2 isolates from NY, 1 from Flinttreatedplants in GA, 4 from Bayleton-treated plants inGA, and 2 from NC were sensitive to both chemicalclasses.Although isolates were not tested from commercialproduction fields, it is prudent for growers to considerimproving their resistance management program.Using strobilurin fungicides alone, as was done in theresearch fields, exerts more selection pressure forstrobilurin resistance than using them with DMIs andcontact fungicides in a resistance managementprogram; however, the size of the population exposedto this high selection pressure in research fields isextremely small compared to that in commercial fields.Strobilurin resistance likely occurs in commercialfields, but was more easily detected in research fieldswhere plants treated with effective fungicides and nontreatedplants provided comparisons. Strobilurinresistance appears to be widespread in the US. It wasconfirmed in GA, VA, NC, and NY. It is likely in AZand CA. And efficacy of strobilurins was reduced insome mid-western states. The cucurbit powderymildew fungus produces spores wind-dispersed overlarge areas. Inoculum for powdery mildew developingon cucurbit crops is thought to be wind-dispersednorthwards through the eastern and mid-western USeach year. Occurrence of resistance in commercialfields will reduce the utility of strobilurins, includingthose not yet registered, and eliminate an importanttool for managing DMI resistance. Strobilurins andDMIs are the only systemic fungicides registered forcucurbit powdery mildew in the US. Currentrecommendations for managing fungicide resistanceinclude using a diversity of fungicides within an71

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2integrated disease management program that includesnon-chemical practices, such as use of resistantcultivars (McGrath, 2001). Nova should be used at themanufacturer's highest label rate (full rate) and shortestapplication interval. One suggested change to improveresistance management is to apply a contact fungicidewith strobilurins as well as DMIs. Sulfur (MicrothiolDisperss®) and mineral oil (JMS Stylet-oil®) arerecommended for resistance management because theyare more effective than Bravo and other contactfungicides for powdery mildew on the lower leafsurface (McGrath 2002a). Quadris applied inalternation with Nova and Microthiol Disperss wasmore effective than Quadris alternated with Nova andBravo in 2002 (Table 1). Sulfur is very inexpensive,but can be phytotoxic to melon (McGrath 2002a).This is the first report of resistance inPodosphaera xanthii to this group of fungicides in theUS. Resistance has already developed in Didymellabryoniae, which causes gummy stem blight, in the US(Stevenson et. al. 2002).REFERENCESAlexander, S. A., and Waldenmaier, C. M. 2002. Evaluation of fungicidesfor control of powdery and downy mildew in pumpkin, 2001.Fungicide and Nematicide Tests 57:V077.Drennan, J. L., and 2000. Comparing fungicides for powdery mildew andgummy stem blight control in butternut squash, 1999. Fungicideand Nematicide Tests 55:259.Everts, K. L., and Armentrout, D. K. 2002. Evaluation of fungicides forcontrol of powdery mildew and Plectosporium blight of pumpkin,2001. Fungicide and Nematicide Tests 57:V080.Hausbeck, M. K., Wendling, N.J., and Linderman, S.D. 2002. Evaluationof fungicides for managing powdery mildew of pumpkin, 2001.Fungicide and Nematicide Tests 57:V081.Ishii, H., Fraaije, B. A., Sugiyama, T., Noguchi, K., Nishimura, K.,Takeda, T., Amano, T., and Hollomon, D. W. 2001. Occurrence andmolecular characterization of strobilurin resistance in cucumberpowdery mildew and downy mildew. Phytopathology 91:1166-1171.Langston, D. B., and Kelley, W. T. 2002. Evaluation of fungicides andbiological control materials for control of powdery mildew intransgenic yellow crookneck squash, 2001. Fungicide andNematicide Tests 57:V095.Matheron, M. E., and Porchas, M. 2003. Evaluation of foliar fungicidesfor control of powdery mildew on muskmelon in Yuma, AZ, 2002.Fungicide and Nematicide Tests 58: (accepted for publication).McGrath, M. T. 1996a. Increased resistance to triadimefon and tobenomyl in Sphaerotheca fuliginea populations following fungicideusage over one season. Plant Disease 80:633-639.McGrath, M. T. 1996b. Successful management of powdery mildew inpumpkin with disease threshold-based fungicide programs. PlantDisease 80:910-916.McGrath, M.T. 2000. Evaluation of fungicide programs for managingpowdery mildew of pumpkin, 1999. Fungicide and NematicideTests 55:253-254.McGrath, M. T. 2001. Fungicide resistance in cucurbit powdery mildew:Experiences and challenges. Plant Disease 85(3):236-245.McGrath, M. T. 2002a. Alternatives to the protectant fungicidechlorothalonil evaluated for managing powdery mildew ofcucurbits. Pages 213-221 in: Proceedings of Cucurbitaceae '02:Evaluation and enhancement of cucurbit germplasm, D. Hopkins,ed.McGrath, M. T. 2002b. Evaluation of fungicide programs for managingpowdery mildew of pumpkin, 2001. Fungicide and NematicideTests 57:V86.McGrath, M.T., and Shishkoff, N. 1999. Evaluation of fungicideprograms for managing powdery mildew of pumpkin, 1998.Fungicide and Nematicide Tests 54:230-231.McGrath, M.T., and Shishkoff, N. 2000. Evaluation of fungicideprograms for managing powdery mildew of muskmelon, 1999.Fungicide and Nematicide Tests 55:176-177.McGrath, M.T., and Shishkoff, N. 2001. Evaluation of fungicideprograms for managing powdery mildew of pumpkin, 2000.Fungicide and Nematicide Tests 2001:V76.McGrath, M. T., and Shishkoff, N. 2003. Evaluation of fungicideprograms for managing powdery mildew of pumpkin, 2002.Fungicide and Nematicide Tests 58: (accepted for publication).McGrath, M. T, Staniszewska, H., Shishkoff, N., and Casella, G. 1996.Fungicide sensitivity of Sphaerotheca fuliginea populations in theUnited States. Plant Disease 80:697-703.Olaya, G., Moreno, M., Lum, B., and Heaney, S. Azoxystrobin resistancemonitoring study for Sphaerotheca fuliginea populations collectedin the United States and Mexico. Phytopathology 90:S57, 2000.Stevenson, K. L., Langston, D. B., and Seebold, K. 2002. Resistance toazoxystrobin in the gummy stem blight pathogen in Georgia.Resistant Pest Management 12(1).Turini, T.A. 2002 Comparison of fungicides for control of powderymildew on muskmelon, 2001. Fungicide and Nematicide Tests57:V050.Turini, T.A. 2003 Comparison of fungicides for control of powderymildew on muskmelon, 2002. Fungicide and Nematicide Tests 58:(accepted for publication).Zitter, T.A., and Drennan, J. L. 2001. Comparing fungicides for powderymildew and gummy stem blight control in butternut squash, 2000.Fungicide and Nematicide Tests 56:V91.Margaret T. McGrath and Nina ShishkoffDepartment of Plant PathologyCornell University Long Island Horticultural Research and ExtensionCenter3059 Sound Avenue, Riverhead, NY 11901-1098United StatesBaseline Sensitivity of Cucurbit Powdery Mildew (Podosphaera xanthii) to the Fungicide Azoxystrobin in theUnited StatesINTRODUCTION Azoxystrobin is the first syntheticfungicidal compound derived from naturally occurringstrobilurins (Ypema and Gold 1999). It is in fungicidegroup 11, the quinone outside inhibitor (QoI) activitygroup. The purpose of this study was to examinebaseline sensitivity of Podosphaera xanthii toazoxystrobin in the United States.MATERIALS and METHODS Isolates of Podosphaeraxanthii were obtained from six states in 1996 (Table 1).A reference isolate collected in 1990 was also included.A leaf-disk bioassay was used to determinesensitivity to azoxystrobin (McGrath et al 1996). Twoweekold squash seedlings ('Seneca Prolific') weresprayed with active ingredient (azoxystrobin 96%)dissolved in methanol: acetone: water (1:1:2 v:v:v) at0, 0.25, 0.5, 1.0, 2.5, and 5 µg/ml. Test solutions were72

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2sprayed onto plants using aDeVilbiss bottle attached to acompressed air source (20 psi).Treated plants were allowed to dry,then disks were cut from thecotyledons using a #9 cork borer (9mm diameter). Five disks of eachtreatment were placed on water agarin divided petri plates (threetreatments per plate). Disks wereinoculated by transferring 5-10conidia to the center of each disk. Ineach trial, duplicate sets of treateddisks were inoculated with eachmildew isolate, then plates wereincubated for approximately 2 weeksat 24 C, at which time the controltreatment showed good growth, withsporulating mildew covering anaverage of 40-60% of leaf disk area.For each fungicide concentration,growth of the mildew was considered to have occurredif sporulation was observed on 3 out of 5 disks (or 2out of 4, if a disk died during incubation). The percentleaf disk area colonized by sporulating mildew wasrecorded for each disk and averaged for each treatment.RESULTS and DISCUSSION Seventy-two powdery mildewisolates from six states showed little variation insensitivity to azoxystrobin. All were able to grow at0.25 µg/ml, 60% could grow at 0.5-1 µg/ml but nohigher, 35% grew slightly (0.4-11% disk areacolonized) on leaf disks treated at 2.5 µg/ml, and 6% (4isolates) grew slightly (0.2-2.8%) at 5 µg/ml. Some ofthe variation in fungus growth may have been due tovariation in the actual concentration of test solutionsused to spray test plants; the fungicidal activeingredient was not readily soluble in water at 5 µg/mland took some time to dissolve in anacetone:methanol:water (1:1:2) solution. It wasnecessary to dissolve the fungicide in acetone:methanol(1:1) and then add water. There was no evidentcorrelation between sensitivity and geographic locationor sensitivity and race. An isolate with resistance totriadimefon and benomyl (isolate 4at) was unable togrow on disks treated with >1 µg/ml azoxystrobin,indicating that resistance to triazoles andbenzimidazoles was independent of resistance toazoxystrobin.REFERENCESMcGrath, M.T., H. Staniszewska, and N. Shishkoff, 1996. Fungicidesensitivity of Sphaerotheca fuliginea populations in the UnitedStates. Plant Disease 80:697-703.Ypema, H. L. and Gold, R. E. 1999. Kresoxim-methyl: Modification of anaturally occurring compound to produce a new fungicide. PlantDisease 83:4-19.Nina Shishkoff and Margaret T. McGrathDepartment of Plant PathologyCornell University Long Island Horticultural Research and ExtensionCenter3059 Sound AvenueRiverhead, New York 11901-1098United StatesManaging Phenylamide Resistance in Potato Late Blight in Northern IrelandINTRODUCTION Formulations containing phenylamides+ mancozeb were approved for the control of potatolate blight in the UK in 1978 and rapidly becamewidely used. In summer 1980, in the Republic ofIreland metalaxyl alone failed to control the diseaseand isolates of Phytophthora infestans from the foliagewere found to be phenylamide-resistant (Dowley andO'Sullivan, 1981). In Northern Ireland, phenylamideresistantisolates of P. infestans were obtained fromblighted tubers from the 1980 crop and annual surveysof the incidence of phenylamide resistance wereinitiated starting in 1981 (Cooke, 1981). In the early1980s, in Northern Ireland the percentage of isolatescontaining phenylamide-resistant strains was generally10-20% (except in 1984), but in the late 1980s therewas a dramatic increase to c. 90% in 1987-89. This wasattributed to the selection pressure resulting fromwidespread and season-long use of formulationscontaining phenylamides + mancozeb (mainlymetalaxyl + half-rate mancozeb) and a succession ofvery wet summers which favoured late blight. Asimilar build-up of phenylamide-resistant strains73

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2occurred in the Republic of Ireland, GreatBritain, and the Netherlands. In 1988, themetalaxyl + mancozeb formulation waschanged to give a full-rate of mancozeb. Inthe early 1990s, an anti-resistance strategybased on the one developed in theRepublic of Ireland (Dowley et al., 1995)was adopted in Northern Ireland withgrowers advised to use no more than threeapplications of phenylamides at thebeginning of the spray programme only.MATERIALS and METHODS Samples ofinfected potato foliage were obtained(mainly from seed crops) by members ofthe Department of Agriculture and RuralDevelopment (DARD) Potato InspectionService (Cooke et al., 2000). Isolates werederived by bulking together the sporangiaobtained from all foliage samples within asingle crop and maintained on detachedglasshouse-grown potato leaflets thentested, using the floating leaf disctechnique (Cooke, 1986), on 100 and 2 mgmetalaxyl litre-1. Isolates were designatedresistant if they sporulated on 100 mgmetalaxyl litre-1-treated discs andsensitive if they sporulated on untreateddiscs, but not on any metalaxyl-treateddisc. Isolates that grew on discs floating on2 mg, but not on 100 mg metalaxyl litre-1,were designated intermediate. At the endof each season, inspectors providedestimates of fungicide usage for all seedpotato crops in their areas.RESULTS and DISCUSSION In the mid-1980s,a high proportion of Northern Irelandpotato crops were sprayed withphenylamide fungicides for most of thespray programme. However, during mostof the 1990s and up to 2002, PotatoInspectors' estimated that c. 40% of seedpotato crops were phenylamide-treated(Fig. 1) and the majority of growersfollowed DARD advice and used no morethan three phenylamide applications perseason (Table 1). The proportion ofisolates containing phenylamide-resistantstrains declined in the early 1990s andappeared to have stabilised around 50%(Fig. 2) up to 1999. However, in 2000 andparticularly in 2001, there was a markedincrease in the incidence of phenylamideresistance. The reason for this was notclear, since fungicide usage had notchanged. The weather in the summermonths of 1998-2000 was unusually wet74

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2and it is possible that this may have increased thenumber of generations of P. infestans within eachseason and favoured the buildup of resistant strains(Fig. 3). In 2002, DARD and Syngenta agreed revisedrecommendations for phenylamide usage: growerswere advised to use no more than two applications perseason and to switch to an alternative product type nolater than 15 July. Subsequently, the proportion ofisolates containing resistant strains declined from 76%in 2001 to 60% in 2002. These growerrecommendations will be continued in 2003 and theincidence of resistant strains again monitored. It isconcluded that in a region such as Northern Ireland,where fit phenylamide-sensitive and -resistant strainsof P. infestans co-exist, resistance may be managed bya strategy of limited use of phenylamides early in theseason only. During the winter period when thepathogen survives in infected tubers, more resistantthan sensitive strains tend to be lost by tuber rottingand this helps to stabilise the situation (Walker andCooke, 1990). However, in regions where aggressivephenylamide-resistant strains have been introduced bymigration rather than in situ selection, as occurredrecently in Taiwan (Deahl et al, 2002), such resistancemanagement may not be possible.REFERENCESCooke, L.R. 1981. Resistance to metalaxyl in Phytophthora infestans inNorthern Ireland. Proceedings 1981 British Crop ProtectionConference - Pests and Diseases 2:641-649.Cooke, L.R. 1986. Acylalanine resistance in Phytophthora infestans inNorthern Ireland. Proceedings of the 1986 British Crop ProtectionConference - Pests and Diseases 2:507-514.Cooke, L.R., Little, G., Wilson, D.G. and Thompson, D. 2000. Up-dateon the potato blight population in Northern Ireland - fungicideresistance and mating type. Fourth Workshop of an EuropeanNetwork for Development of an Integrated Control Strategy ofpotato late blight. Oostende, Belgium, 29 September - 2 October1999. PAV-Special Report no. 6, 35-45.Deahl, K.L., Cooke, L.R., Black, L.L. Wang, T.C., Perez, F.M., Moravec,B.C., Quinn, M. and Jones, R.W. 2002. Population changes inPhytophthora infestans in Taiwan associated with the appearance ofresistance to metalaxyl. Pest Management Science 58:951-958.Dowley, L.J., Cooke, L.R. and O'Sullivan, E. (1995). Development andmonitoring of an anti-resistance strategy for phenylamide useagainst Phytophthora infestans. In Phytophthora infestans. pp. 130-136. Eds.Dowley, L.J., Bannon, E., Cooke, L.R., Keane, T. & O'Sullivan, E. BoolePress, Dublin. 382 pp. Dowley, L.J. and O'Sullivan, E. 1981.Metalaxyl-resistant strains of Phytophthora infestans (Mont.) deBary in Ireland. Potato Research 28:417-421.Walker, A.S.L. and Cooke, L.R. 1990. The survival of Phytophthorainfestans in potato tubers - the influence of phenylamide resistance.Proceedings Brighton Crop Protection Conference - Pests andDiseases - 1990 3:1109-1114.Louise R. CookeApplied Plant Science DivisionDepartment of Agriculture and Rural DevelopmentNewforge Lane Belfast, BT9 5PXUnited KingdomResearch in Resistance ManagementActivity Spectrum of Spinosad and Indoxacarb: Rationale for an Innovative Pyrethroid Resistance ManagementStrategy in West AfricaABSTRACT To face pyrethroid resistance in the cottonbollworm Helicoverpa armigera (Hübner), endosulfan(700 g/ha) has been used in a resistance managementstrategy for four years in Côte d'Ivoire, West Africa.Actually, its recommendation is being questioned withregard to its acute toxicity and environmental issues.Earlier prospects revealed that insecticides such asspinosad (48 g/ha) and indoxacarb (25 g/ha) proved aseffective as endosulfan in controlling H. armigera. Incontrast to endosulfan, the activity spectrum of thesenon-pyrethroids insecticides appears to be restricted toa few bollworm and leaf pests. The present studypointed out the strength and weakness of these newinsecticides with respect to major insect pests andbeneficial species. On the basis of their activityspectrum and in the light of cotton crop phenology andmain pest seasonal occurence, a differential schemewas designed. Indoxacarb is more appropriate to thefruiting stage (101-115 DAE (Day After Emergence))as it appeared very effective against the cotton stainerDysdercus voelkeri (Schmidt) while showing lowerperformance against Earias spp and the mitePolyphagotarsonemus latus (Bank). In contrast,spinosad is to be used preferably at the vegetative stage(45-66 DAE) as it proved safer to coccinellids, moreeffective against Earias spp while its lowereffectiveness against D. voelkeri suggests avoiding itspositioning at a late stage of cotton. Various benefitsrelated to these new insecticides strongly advise theiruse as alternatives to pyrethroids. Still, to be moreattractive, their activity needs to be reinforced by otherinsecticides in such a way to control the wholearthropod pest complex.KEY WORDS: Cotton, Helicoverpa armigera, pyrethroidresistance management strategy, Spinosad, Indoxacarb,Côte d'Ivoire.INTRODUCTIONThe development of resistance in H. armigera:Known as very effective in controllingHelicoverpa armigera (Hübner) and most cottonbollworm pests, pyrethroids have been widely used formore than twenty years in Côte d'Ivoire. Recently,laboratory data obtained on H. armigera strains within1996-1998 pointed out significant increase in the LD5075

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2for both deltamethrin (Figure 1) and cypermethrin(Vassal et al., 1997; Vaissayre et al., 1998; Martin etal., 2000). Field data recorded for eight consecutiveyears (Figure 2) revealed that the pest infestationprofiles changed deeply from 1991 to 1998 (Ochou etal., 1998). Moreover, cases of ineffectiveness of thepest control programme against H. armigera have beenreported during exceptional pest outbreaks in Côted'Ivoire. With this regard, the routine calendar-basedprogramme applying six fortnightly sprays ofpyrethroid-organophosphate insecticide mixtures overthe whole cotton season has been questionned as thepyrethroid resistance in H. armigera was confirmed(Ochou & Martin, 2000). Similar cases of resistancewere reported in H. armigera in most West Africancountries (Benin, Burkina Faso, Guinea, Mali, Senegal,and Togo) (Anonymous, 1999).Development of the IRM strategy against H. armigera:To face pyrethroid resistance in the cottonbollworm, H. armigera, an Insect ResistanceManagement (IRM) programme, inspired from the"Australian" strategy (Sawicki and Denholm, 1987),was designed in Côte d'Ivoire. In practice, the strategyhas led to the determination of a pyrethroid-free seasonnationwide by using non-pyrethroid insecticides(endosulfan 700-750g/ha and profenofos 750 g/ha) in akind of "window" programme in order to lessenpyrethroid selection pressure. The pyrethroid-freeseason is established according to cotton growingzones (August 10 and August 20 respectively fornorthern and southern regions). The main picturewhich has come out from the nationwide adoption ofthe pyrethroid resistance management programme bycotton farmers is the important decrease in the fieldpopulations of H. armigera (Figure 2) since 1998(Ochou & Martin, 2002).Endosulfan has been widely used in the currentpyrethroid resistance management programme over thelast four years in Côte d'Ivoire, and so far, no resistanceto endosulfan has been detected (Martin et al., 2002).However, its recommendation is being actuallyquestioned with regard to its toxicity, environmentalissues, and farmer practices. To tackle this problem,investigations are being undertaken to adapt arelatively low dose of endosulfan (525 g/ha) to theactual field infestation of H. armigera (Ochou &Martin, 2000) and to assess microencapsulatedformulations of endosulfan, assumed safer than the ECformulations. At the same time, further investigationsfocused on new insecticides such as spinosad andindoxacarb as potential alternatives to endosulfan.Spinosad is a naturally produced mixture of theactinomycete Saccaropolyspora spinosa. Its mode ofaction is described as an activation of the nicotineacetylcholine receptor, but at a different site fromnicotine or imidachloprid. It is activated by contact andingestion, causing paralysis (Pesticide Manual, 12thedition, v2). Indoxacarb is an oxadiazine product wherethe active component blocks sodium channels in nervecells. It is activated by contact and ingestion, andaffected insects cease feeding, with poor co-ordination,paralysis, and ultimately death (Pesticide Manual, 12thedition, v2). Due to their novel mode of action, bothinsecticides appear ideal for resistance managementprogrammes. However, to be rationally used, there is aneed for a precise activity spectrum of these newinsecticides that proved as effective as endosulfan incontrolling H. armigera (Ochou & Martin, 2002).The present study is to assess the activity spectrumof spinosad and indoxacarb with regard to beneficialsand major components of the cotton pest complex inCôte d'Ivoire. The need to reinforce their activity byother insecticides will be also assessed. On the basis ofthe strength and weakness of these new insecticidesand with respect to cotton crop phenology and seasonaloccurence of main pests, appropriate recommendationswill be stated to justify their integration into thepyrethroïd resistance management programmes.76

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2MATERIALS and METHODS The study was carried out forthree consecutive years (1999-2001) at the cottonresearch station of CNRA based at Bouaké and at theexperimental station of LCCI at Nambingué. At first,the biological activity of the two specific insecticides(spinosad 48g/ha (Laser 480 SC, Dow AgroSciences)and indoxacarb 25g/ha (Avaunt 150 SC, Du Pont)) wasassessed in reference with endosulfan 750 g/ha (Phaser375 EC, Aventis), and deltamethrin 12 g/ha (Decis 12EC, Aventis) through a Complete Bloc Design with sixreplicates. Individual plots were of 10 rows x 15 m.Further field trials were undertaken in a similar designwith the two insecticides in association with otherinsecticides. Tested mixtures included spinosad 48g/ha+ profenofos 300g/ha, spinosad 48g/ha + acetamiprid10g/ha, indoxacarb 25g/ha + profenofos 300g/ha,indoxacarb 25g/ha + acetamiprid 10g/ha, andcypermethrin 36g/ha + profenofos 300g/ha.Insecticides sprays were performed with anadapted horizontal boom knapsack sprayer debiting 60l/ha of product-water mixture. Plots were treated every14 days from 45th to 115th DAE (day after emergenceof cotton). Fields were scouted directly on plants oncea week from 30th to 122nd DAE for sucking pests,leafworms, and exocarpic bollworms pests, and everytwo weeks on green bolls from 70th to 112th DAE forendocarpic bollworms. Target pests and beneficialswere recorded as follows:1. mite Polyphagotarsonemus latus infested plants p.3 rows x 15m;2. aphid Aphis gossypii infested plants p. 3 rows x15m;3. jassid Jacobiella fascialis infested plants p. 30plants;4. individual sucking pests (Dysdercus voelkeri,Bemisia tabaci), leafworms (Spodoptera littoralis,Anomis flava, Syllepte derogata), and exocarpicbollworms (H. armigera, Earias spp, Diparopsiswatersi) p. 30 plants;5. endocarpic bollworms (Cryptophlebia leucotreta,Pectinophora gossypiella) p. 100 green bolls; and6. individual beneficials (ladybirds, spiders, etc.) p.30 plants.Three year average data for all bollworms and onetwoyear average data for sucking pests, leaf pests, andbenefials were considered.RESULTSEffectiveness of spinosad and indoxacarb againstcotton bollworms:Data presented in Figures 3a-d show comparedeffectiveness of the pyrethroid deltamethrin and thenon pyrethroïd insecticides on cotton exocarpicbollworm species (H. armigera, Earias spp., D.watersi) and endocarpic bollworm species (C.leucotreta and P. gossypiella).Spinosad activicty on the exocarpic bollwormspecies was at least equivalent to endosulfan as areference: H. armigera (3.1 vs 3.4 larvae p. 30 plants),Earias spp, and D. watersi. Overall activity of spinosadagainst the exocarpic bollworm species was higher thandeltamethrin. Indoxacarb activity was at the level ofdeltamethrin for H. armigera (4.9 vs 5.1 larvae p. 30plants), and to a certain extent less effective againstEarias spp. In contrast, the activity of both insecticides(spinosad and indoxacarb) on endocarpic speciesremained low in relation to deltamethrin (6.4 and 7.1 vs3.2 endocarpic larvae p. 100 bolls, respectively forspinosad, indoxacarb, and deltamethrin).Effectiveness of spinosad and indoxacarb againstsucking pests:Data presented in Figures 4a-d reveal comparedactivity of the pyrethroïd deltamethrin and the nonpyrethroïd insecticides on cotton sucking pests J.fascialis, A. gossypii, D. voëlkeri, and the mite P. latus.The effect of spinosad was at least equivalent todeltamethrin on the jassid J. fascialis (1.2 vs 1 jassidattacked plants p. 30 plants) and on the mite P. latus (4mite infested plants p. 3 rows). In contrast, spinosadappeared less effective than endosulfan against theaphid A. gossypii (56.8 vs 36.8 aphid infested plants p.3 rows) and the cotton stainer D. voëlkeri (169 vs 140.8Dysdercus p. 30 plants). Contrary to spinosad, theeffect of indoxacarb was equivalent to deltamethrin onD. voëlkeri (110.3 vs 101.8 Dysdercus p.30 plants) andon the aphid A. gossypii (43.3 vs 48.8 aphid infestedplants p. 3 rows) while showing less effectivenesscompared to endosulfan against the mite P. latus (11.5vs 2.4 mite infested plants p. 3 rows).Effectiveness of spinosad and indoxacarb againstcotton leafworms:Data presented in Figures 5a-b show the comparedeffect of the pyrethroid deltamethrin and the nonpyrethroïd insecticides on cotton leafworm S. littoralisand A. flava.Spinosad and indoxacarb proved very effectiveagainst the leafworm S. littoralis (0.7 and 0.8 vs 1.5larvae p. 30 plants, respectively for indoxacarb,spinosad, and deltamethrin). Their activity of on A.flava remained equivalent to deltamethrin andendosulfan (1.2 and 2.2 vs 1.8 larvae p. 30 plants,respectively for spinosad, indoxacarb, and endosulfan).Activity of spinosad and indoxacarb on beneficials:Figures 6a-b show data on the compared activityof the pyrethroïd deltamethrin and the non pyrethroïdinsecticides on beneficial predators.Spinosad and indoxacarb to a lesser extent provedsafer on ladybirds, Coccinela spp., as compared toendosulfan (10.7 and 5.8 respectively for spinosad and77

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2indoxacarb vs 4 coccinellids p.30 plants). The effect ofboth insecticides on the spiders was equivalent toendosulfan (6.5 vs 7 spiders p.30 plants).Effectiveness of spinosad and indoxacarb in mixturewith other insecticides:Data presented in Figures 7a-d showed comparedactivity of spinosad or indoxacarb based associationswith profenofos and acetamiprid, and pyrethroïd basedassociations on cotton bollworms and some suckingpests.The profenofos based association with spinosad orindoxacarb provided an activity level at leastequivalent to cypermethrin-profenofos associationagainst H. armigera (0.3 and 1 vs 1.1 larva p. 30plants, respectively for indoxacarb-profenofos,spinosad-profenofos and cypermethrin-profenofos).The same tendency was observed against the mite P.latus (0.1 and 2.5 vs 2.9 mite infested plants p. 3 rows).The acetamiprid-based association with spinosadwas at least equivalent to the cypermethrin-acétamipridassociation against D. voëlkeri (74.2 vs 90.7 Dysdercusp. 30 plants). This association was much more effectiveagainst D. voelkeri than the indoxacarb-acetamipridassociation (109.3 Dysdercus p. 30 plants). Concerningthe endocarpic bollworm species (C. leucotreta & P.gossypiella) the spinosad-acetamiprid associationshowed an activity level equivalent to thecypermethrin-acetamiprid (4 vs 2 larvae p. 100 bolls)while the activity remained very low for theindoxacarb-acetamiprid association (9.5 larvae p. 100bolls).DISCUSSION The present study pointed out the strengthsand weaknesses of spinosad and indoxacarb withrespect to major insect pests and beneficial species.The activity of spinosad and indoxacarb variedsignificantly according to insect pest species orbeneficial species.The spinosad activity spectrum comprisedexocarpic bollworm species (H. armigera, Earias spp,and D. watersi) and cotton leafworms S. littoralis andA. flava. It appeared to have a certain activity againstthe endocarpic bollworm species (C. leucotreta & P.gossypiella), the jassid J. fascialis, and the mite P.latus. This activity noticed especially on sucking pestssuch as the jassid J. fascialis and the mite P. latus needto be confirmed in more field trials, for the manualpesticide states that spinosad is non toxic to suckingpests. Indeed, spinosad appeared very limited againstthe aphid A. gossypii and the cotton stainer D. voëlkeri.With regard to benefials, spinosad proved safer toCoccinela spp and spiders.79

In contrast to spinosad, indoxacarb activityspectrum was restricted to a few bollworm species (H.armigera, D. watersi) and the cotton leafworm S.littoralis. In addition, it appeared to have a certaineffectiveness against the jassid J. fascialis, the aphid A.gossypii, and the cotton stainer D. voëlkeri. Indoxacarbappeared somehow inactive on Earias spp., the mite P.latus, and the endocarpic bollworm species (C.leucotreta & P. gossypiella).On the basis of their activity spectrum and in thelight of cotton crop phenology and seasonal occurenceof main pests, differential pyrethroïd resistantmanagement plans could be designed (Figures 8a-b)considering the positioning of spinosad and indoxacarbeither at the vegetative or fruiting stages of cotton.Due to its high effectiveness on exocarpicbollworm species mainly H. armigera and Earias sppand its relative safety to major beneficials such asladybird Coccinela spp, spinosad could be usedpreferably at cotton vegetative stage (45-66 d.a.e.). Therelatively broad activity spectrum of spinosad makes itideal for use at the vegetative stage of cotton,appearing as a true alternative to endosulfan. Itspositioning at the late stage of cotton could be moresuitable provided it would be used in association withother insecticides such as acetamiprid, effective againstD. voelkeri and A. gossypii. Due to its activity

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2spectrum, which is relatively restricted in relation tospinosad, indoxacarb appears more appropriate to thecotton fruiting stage (101-115 d.a.e), as it provedeffective against the cotton stainer D. voelkeri whileshowing lower performance against Earias spp and themite P. latus. Association of indoxacarb with otherinsecticides such as profenofos could enhance itsactivity mainly on the mite P. latus. The use ofindoxacarb is not advisable during the period thatcoincides with maximum flowering for it had a limitedeffect on endocarpic bollworm species (C. leucotretaand P. gossypiella) which occur in largest numbers atthis stage; it is therefore necessary to maintain apyrethroid based association in order to controlendocarpic bollworm species.Various benefits related to these new insecticidesstrongly advise their use as alternatives to pyrethroids.Still, to be more attractive, their activity needs to bereinforced by other insecticides in such a way as tocontrol the whole arthropod pest complex. Conjoinedlaboratory activities are being achieved to help setmore reliable strategies and improve the whole pestmanagement strategy. Bioassays performed withseveral classes of insecticides, especially nonpyrethroïd insecticides such as DDT, endosulfan,profenofos, indoxacarb, and spinosad did not show anycross-resistance with pyrethroids in H. armigera(Martin, unpublished data), knowing that pyrethroidresistance in H. armigera from West Africa was due togreater degradation of pyrethroids involving oxidasesfrom the P450 family (Martin et al., 2002).CONCLUSION The earlier use of endosulfan andprofenofos as pyrethroid alternatives in H. armigeraresistance management in Côte d'Ivoire helpedsubstantially reduce field infestations of H. armigerafor the last four years. No resistance was detected forendosulfan and profenofos in field populationsindicating the success of these pyrethroid alternatives.However, endosulfan and profenofos resistance wasshown in H. armigera from Pakistan (Ahmad et al.,1995) and Australia (Forrester et al., 1993; Gunning etal., 1993) indicating the risk to select resistant larvae inCôte d'Ivoire if those insecticides are to be used foryears without alternatives. For the pyrethroid resistancemanagement to be sustainable, there is a clear need toadopt alternative insecticides such as spinosad andindoxacarb in a rational non pyrethroid insecticiderotation plan. Spinosad and indoxacarb could be usedin appropriate resistance management programmeseither alone or reinforced in mixture by otherinsecticides or in mosaic with endosulfan andprofenofos in such a way to avoid the selection of newcases of resistance.ACKNOWLEDGEMENTS The authors acknowledge theresearch and development staff of cotton companies ofCôte d'Ivoire (CIDT, IC, LCCI) and MM. Konan K.Jérôme, Kouadio René, and Kouadio Gérard of thecotton entomology technical research team of CNRAfor their assistance in collecting field data. Thanks aredue to chemical companies Dow AgroScience, Du Pontde Nemours, Aventis CropScience, and Syngenta forinsecticide samples provided.REFERENCESAhmad, M., Arif, M.I., Ahmad, Z. 1995. Monitoring insecticideresistance of Helicoverpa armigera (Lepidoptera Noctuidae) inPakistan. Journal of Economic Entomology. 88 (4): 771-776.Forrester, N.W., Cahill, M., Bird, L. J. and Layland, J. K. (1993)Management of pyrethroid and endosulfan resistance inHelicoverpa armigera (Lepidoptera: Noctuidae) in Australia. InBull. Entomol. Res. Supplement Series N°1. International Instituteof Entomology pp. 1-132.Gunning, R. V. and Easton, C. S. (1993). Resistance to organophosphateinsecticides in Helicoverpa armigera (Hübner) (Lepidoptera:Noctuidae) in Australia. General Applied Entomology, 25: 27-34.Martin, T., Ochou, G.O., Hala, N.F., Vassal, J.M. and Vaissayre, M.(2000). Pyrethroid resistance in the cotton bollworm, Helicoverpaarmigera (Hübner), in West Africa. Pest Management Science, 56,549-554.Martin, T., Chandre, F., Ochou, O. G., Vaissayre, M. and Fournier, D.(2002). Pyrethroid resistance mechanisms in the cotton bollwormHelicoverpa armigera (Lepidoptera: Noctuidae) from West Africa.Pesticide Biochemical Physiology, 74 : 17-26.Martin, T., Ochou, O. G., Vaissayre, M. and Fournier, D. (2002). Positiveand Negative Cross-resistance to Pyrethroids in Helicoverpaarmigera from West Africa. Resistant Pest ManagementNewsletter, 12, 1.Ochou, O.G., Martin, T. and Hala, N.F (1998). Cotton insect pestproblems and management strategies in Côte d'Ivoire, W. Africa.Proceedings of the world Cotton Research Conference-2. Athens,Greece, September 6-12, pp 833-837.Ochou, O.G. and T. Martin (2000). Prévention et gestion de la résistancede Helicoverpa armigera (Hübner) aux pyréthrinoïdes en Côted'Ivoire. 2ème Rapport d'exécution technique du projet régional PR-PRAO. 93 p.Ochou, G. O. and Martin, T. (2002). Pyrethroïd Resistance inHelicoverpa armigera (Hübner): Recent Developments andProspects for its Management in Côte d'Ivoire, West Africa.Resistant Pest Management Newsletter, 12, 1.Sawiki, R.M. and I. Denholm (1987). Management of resistance topesticides in cotton pests. Tropical Pest Management 33 (4):262-272.Vaissayre, M., Vassal, J. M. and Martin, T. (1998). Pyrethroid resistancein the bollworm Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae) in West Africa. Proceedings of the World CottonResearch Conference-2. Athens, Greece, September 6-12, pp. 701-705Vassal, J.M., M. Vaissayre and T. Martin (1997). Decrease in thesusceptibility of Helicoverpa armigera (Hübner) (Lepidoptera :Noctuidae) to pyrethroid insecticides in Côte d'Ivoire. ResistantPest Management, 9, 2, 14-15.O.G. OchouCentre National de Recherche Agronomique (CNRA)01 BP 633 Bouaké 01Côte d'IvoireT. MartinCentre National de Recherche Agronomique (CNRA)01 BP 633 Bouaké 01Côte d'IvoireCentre de Coopération International en Recherche Agronomique(CIRAD)34398 MontpellierFrance81

Resistance Management NewsInsect Molecular GeneticsMarjorie A. HoyDepartment of Entomology & NematologyUniversity of FloridaP.O. Box 110620 Gainesville, FL 32611-0620The second edition of Insect Molecular Genetics, ISBN0-12-357031-X, is now available from AcademicPress/Elsevier for $79.95.It can be ordered at: 1-800-545-2522 (USA) or atwww.elsevier.com (USA) or www.elsevierinternational.com.WAHRI Research Results and NewsThe latest research results and news from the WAHerbicide Resistance Initiative (WAHRI) is nowavailable (http://wahri.agric.uwa.edu.au/news.html).In this issue:WEEDEM: A new tool for predicting in-cropweed emergence is now availableWill the world's greatest herbicide continue toaid world food production?Research shows how glyphosate resistance canbe prevented.Low herbicide rates and rapid selection forresistance.The most effective practices for targeting weedseeds at harvest.Annual ryegrass seedbanks: the good, the bad,and the ugly.What value do growers place on glyphosate andis resistance expected?Topactive workshop package now available:Weed & resistance management for long termprofit.WAHRI a partner in WA Grower GroupAlliance.Latest publications.Link to the latest WAHRI News & Views:http://wahri.agric.uwa.edu.au/news.htmlIf you wish to add an e-mail address to the list, pleasesend an e-mail to wahri-news@agric.uwa.edu.au.News & Views is edited by:Mechelle Owen & Rick LlewellynWestern Australian Herbicide Resistance InitiativeUniversity of Western Australiahttp://wahri.agric.uwa.edu.auEffect of Systemic Acquired Resistance on theSusceptibility of Insect Herbivores toEntomopathogensInduced systemic resistance and systemic acquiredresistance in plants involve major biochemical changesresulting in resistance to pathogens, reduced diseaseexpression, and direct effects on herbivores. Tritrophiceffects on the pathogens of herbivorous insects havenot yet been described, however. If an insect feedingAbstractson induced plants is stressed in some manner it may bemore susceptible to pathogens. We are studying threemodel systems to examine these tritrophic effects: thesusceptibility of the orthopteran Melanoplussanguinipes, and the lepidotperans Ostrinia nubilalisand Agrotis ipsilon, all feeding on Systemic AcquiredResistance (SAR)-induced corn plants, to Beauveriabassiana Strain GHA. SAR was induced by applicationof a commercial preparation of harpin. Immatureinsects were reared on induced and corn plants and

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2then bioassayed as young adults (M. sanguinipes) or4th instar larvae (the two lepidopterans) with thefungus.From the Annual Meeting of the EntomologicalSociety of America, Nov. 17-21, 2002, Ft. LauderdaleFL.P. AveryLee AcademyLee, ME 04455S. T. JaronskiUSDA ARS NPARLSidney, MT 59270Kit for the Detection of Echinochloa colona andIschaemum rugosum Susceptibility Status to theHerbicide Fenoxaprop-p-ethylA kit for the detection of the status ofsusceptibility of Echinochloa colona and Ischaemumrugosum to the herbicide fenoxaprop-p-ethyl is a newtool that allows verification of the loss of susceptibilityfrom these two grass weeds to the herbicide in areliable, quick, and simple way. The kit is the result ofseveral years of research in two different laboratoriesworking simultaneously: the Laboratory of Herbicidesof "La Tupia" Bayer Crop Science ExperimentalStation located at Cauca Valley-Colombia and theWeed Science Laboratory of the Agronomy Faculty-National University of Colombia, Bogotá. Previous andexperimental work included more than 60 experimentsand kit validation under field conditions. Initially,different techniques were evaluated by dose-responsebioassays considering seeds, meristems, seedlings(shoots) growing in nutrient solutions, and foliartreatment with a microsyringe, compared with thetraditional methodology of herbicide treatment in awide dose range to plants growing in pots. Twostandards (purified biotypes) - one sensitive and oneresistant - of each one of the two weed species wereconsidered in all experiments. "Seedling (shoots) test"showed the best performance and is therefore the oneon which this kit has been based. To develop the kit,the following were determined: flask size, plant state,time of activity of the herbicide solutions onceprepared, optimal date of visual fitotoxicity testing,DG50, discriminatory dose (dose that marks thebiggest difference between the sensitive standard andthe resistant one), and optimal population sample size.DG50 was calculated by a log-logistic model. The kitmanual instruction explains in a simple and practicalway the procedure of gathering the plants in the field,of seedling assembly in the flasks, and of evaluatingherbicide fitotoxicity. This kit will not be marketed. Itwill have restricted use for the technical staff of BayerCrop Science in Colombia and in any other country.Cilia L. FuentesNational University of ColombiaAgronomy Faculty-BogotáSantiago Montoya, Bernard Jacqmin & NorbertoHernándezBayer Crop ScienceColombiaRelative Susceptibility in Open and GreenhousePopulations of Two-Spotted Spider Mite,Tetranychus urticae Koch, on Rose to DicofolDicofol is a very commonly used acaricide for themanagement of spider mites. Development ofresistance to miticides in spider mites (Tetranychidae)is often so rapid that effective spider mite managementis difficult in many agricultural systems (Jeppson et al.,1975). There are extensive reports worldwide regardingresistance of the two-spotted spider mite, Tetranychusurticae Koch, to different groups of chemicals.However, there is no information available regardingrelative susceptibility of T. urticae to dicofol from openand greenhouse populations from India. In response toan increasing number of treatment failures of dicofolagainst T. urticae on rose, as expressed by farmers inand around Bangalore, this study was taken up.Bioassays were carried out during April-May 2002 toassess the relative susceptibility of open andgreenhouse populations of this mite on rose to dicofol18.5 EC (Kelthane) at the Indian Institute ofHorticultural Research (IIHR), Bangalore. Leaf residuemethod was used for the bioassays as described byFAO (1980). Each concentration was replicated threetimes. Mite mortality was observed 24 h after releaseof mites to the insecticide treated leaves of differentconcentrations. The LC50 values of dicofol to T.urticae were recorded as 0.0404 % for greenhousepopulations and 0.0195 % for open field populations.Thus, greenhouse population of T. urticae haddeveloped 2.1fold resistance to dicofol when comparedto the open field. Relatively more tolerance ofgreenhouse populations of the mite to dicofol might beattributed to more number of generations of mitessubjected to acaricidal sprays when compared to opencultivated roses. The development of cross resistanceto different chemicals frequently used on this cropneeds to be further studied.REFERENCESFAO 1980. Revised method for spider mites and their eggs.FAO MethodNo.10a In: plant production and Protection Paper 21:49-53.Jeppson, L.R., Keifer, H.H. and. Baker. E.W. 1975. Mites injurious toeconomic plants. University of California Press.V. Sridhar and B. Jhansi RaniDepartment of Entomology and NematologyIndian Institute of Horticultural ResearchHessaraghatta LakeP.O., Bangalore-560 089India83

SymposiaCAST Pesticide Resistance Management Symposium Provides Cross-Disciplinary DialogueThe Council for Agricultural Science andTechnology (CAST) held a two-day symposium onApril 10-11, 2003 in Indianapolis, Indiana, entitled"Management of Pest Resistance: Strategies UsingCrop Management, Biotechnology, and Pesticides."CAST is a nonprofit organization composed of 38scientific societies and many individual, student,company, nonprofit, and associate society members.CAST assembles, interprets, and communicatesscience-based information regionally, nationally, andinternationally on food, fiber, agricultural, naturalresource, and related societal and environmental issuesto our stakeholders - legislators, regulators,policymakers, the media, the private sector, and thepublic.This symposium provided a cross-disciplinaryapproach to management of pest resistance and broughttogether 120 professionals concerned with resistancemanagement involving pathogens, insect pests, andweeds. There were representatives from the pesticideindustry, seed companies, extension, academia, stateand federal government (U.S. and Canada), pesticideeducation, consulting agencies, and growerorganizations.The overall goal of the symposium was to providea collective framework in which more proactiveresistance management could be developed in thefuture. The major objectives of the symposium were to:1. identify the common issues related to pesticideresistance management across disciplines;2. identify ways to remove barriers that preventproactive resistance management;3. provide opportunities for future discussions onpesticide resistance management;4. identify future research activities in resistancemanagement; and5. provide this information to lawmakers and federalagencies, especially the United StatesEnvironmental Protection Agency (EPA) and theUnited States Department of Agriculture (USDA),academia, extension, industry, consulting agencies,and the public.Forty-seven speakers from industry, academia,extension, consulting agencies, federal and stategovernment, grower organizations, and public interestgroups gave presentations at the Symposium. Most ofthese presentations will be available on-line at theCAST website (http://www.cast-science.org). Thesymposium agenda was developed by a steeringcommittee consisting of representatives from USDA,EPA, industry (Resistance Action Committees),academia, public interest groups, and growerorganizations. The agenda is available at the CASTweb site. The Symposium was organized into thefollowing eight sessions:1. Scope of North American Pest ResistanceProblems in 20032. Issues in Pest Resistance Management3. Lessons Learned I: Balance between Industry,Academia, Users, and Regulators4. Lessons Learned II: Have Models Helped?5. Role of Stakeholders6. Lessons Learned III: How Can We Work toAlleviate Barriers to Comprehensive ResistanceManagement Implementation? How Can We WorkTogether Better?7. Pest Resistance Management Goals8. Symposium Recommendations for Pest ResistanceManagement - Where to Now?The symposium provided a fruitful opportunity forall stakeholders involved in insect, weed, and pathogenpest management to come together to discuss issuesand lay the foundation for future collaborations toaddress pest resistance management. Several majorinterest areas were explored:1. targeting research funding for pest resistancemanagement with federal competitive grantprograms;2. improving pesticide education programs to addresspest resistance management;3. improving transparency of the EPA's regulation ofpesticide resistance management;4. evaluating of potential economic impacts of pestresistance management;5. targeting consumer education programs on the useof reduced risk pesticides, resistance management,and the cost of producing quality food in themarketplace;6. standardizing of definitions for resistance, methodsof resistance documentation, etc.;7. focusing on goals of resistance monitoringprograms; and8. changing national farm policy to better fundresearch and education for resistance management.Stakeholders agreed that proactive resistancemanagement is a desirable goal, but the path to reachthis goal is unclear.CAST is in the process of developing an on-lineProceedings of the Symposium that should be availableon the CAST website in Fall 2003

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2(http://www.pestmanagement.info/rmworkshop/). Thispublication will be available at no cost to CASTmembers, the media, and government policymakers,and will be accessible to others for a fee. For furtherinformation contact, Sharlene Matten, 202-675-8333,ext. 16 or smatten@cast-science.org.Sharlene R. Matten, Ph.D.On detail to Council for Agricultural Science and Technology (CAST)Science Policy FellowWashington, D.C. [Feb.-June 2003]United StatesandBiologist, U.S. EPAOffice of Pesticide ProgramsWashington, D.C.United StatesDue to lack of subscriber interest and use we willno longer be offering our Perspectives Forum sectionin the newsletter. This site, intended as an onlinediscussion forum to express your views on issuesand/or problems that are of general reader interest, wasnot being utilized. Perhaps if there is interest, thisfeature will be reintroduced in the future.Thank you to those who contributed to this issue -you have really made the newsletter a worthwhilereading experience! Our contributors truely increasethe newsletter's success at sharing resistanceinformation worldwide.We encourage all of our readers to submit articles,abstracts, opinions, etc (see the newsletter online athttp://whalonlab.msu.edu/rpmnews/general/rpm_submission.htm for submission information).Announcements and Submission DeadlinesThe Newsletter is a resource to many around theglobe. It is also a wonderful and effective way toenhance the flow of ideas and stimulate communicationamong global colleagues. We appreciate your efforts tosupport the newsletter and we look forward to yourcontinued contributions.The next two submission deadlines are:Monday, September 15th, 2003Monday, March 15th, 2004We hope you continue to consider the newsletteras a forum for displaying your ideas and research.85

Spring 2003 Resistant Pest Management Newsletter Vol. 12, No.2Libraries that wish to receive a printed version may send a request to:Newsletter CoordinatorResistant Pest Management NewsletterCenter for Integrated Plant SystemsMichigan State UniversityEast Lansing, MI 48824-1311USAPlease visit us online today at http://whalonlab.msu.edu/rpmnews/Editors:Mark E. Whalon (whalon@msu.edu)Robert M. Hollingworth (rmholl@msu.edu)Area Editors: Jonathan Gressel (Herbicide)Margaret Tuttle McGrath (Plant Pathology)Coordinator:Erin Gould (rpmnews@msu.edu)86

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